PHOTODYNAMIC INHIBITION OF MICROBIAL PATHOGENS IN PLANTS

Abstract

There is provided a method for inhibiting growth of a microbial pathogen of a plant. The method includes applying to the plant a combination including a nitrogen-bearing macrocyclic compound which is a singlet oxygen photosensitizer selected from the group consisting of a porphyrin, a reduced porphyrin and a mixture thereof; and a chelating agent to increase permeability of the microbial pathogen to the nitrogen-bearing macrocyclic compound; and exposing the plant to light to activate the nitrogen-bearing macrocyclic compound and generate reactive singlet oxygen species.

Claims

1.-167. (canceled)

168. A method for inhibiting growth of a microbial pathogen of a plant, comprising: applying to the plant a combination comprising: a nitrogen-bearing macrocyclic compound which is a singlet oxygen photosensitizer selected from the group consisting of a porphyrin, a reduced porphyrin and a mixture thereof; and a chelating agent to increase permeability of the microbial pathogen to the nitrogen-bearing macrocyclic compound; and exposing the plant to light to activate the nitrogen-bearing macrocyclic compound and generate reactive singlet oxygen species.

169. The method of claim 168, wherein the chelating agent and the nitrogen-bearing macrocyclic compound are provided in amounts that are synergistically effective to inhibit growth of the microbial pathogen.

170. The method of claim 168, wherein the reduced porphyrin is selected from the group consisting of a chlorin, a bacteriochlorin, an isobacteriochlorin, a corrin, a corphin and a mixture thereof.

171. The method of claim 170, wherein the reduced porphyrin is a chlorin.

172. The method of claim 171, wherein the chlorin is chlorophyllin.

173. The method of claim 168, wherein the nitrogen-bearing macrocyclic compound is complexed with a metal to form a metallated nitrogen-bearing macrocyclic compound, the metal being selected such that, in response to light exposure, the metallated nitrogen-bearing compound generates reactive singlet oxygen species.

174. The method of claim 173, wherein the metal is selected from the group consisting of Mg, Zn, Pd, Al, Pt, Sn, Si and mixtures thereof.

175. The method of claim 168, wherein the nitrogen-bearing macrocyclic compound is a metal-free nitrogen-bearing macrocyclic compound that is selected such that, in response to light exposure, the metal-free nitrogen-bearing compound generates reactive singlet oxygen species.

176. The method of claim 168, wherein the chelating agent comprises an amino polycarboxylic acid compound or an agriculturally acceptable salt thereof.

177. The method of claim 176, wherein the amino polycarboxylic acid compound is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA) or an agriculturally acceptable salt thereof, ethylenediamine-N,N-disuccinic acid (EDDS) or an agriculturally acceptable salt thereof, iminodisuccinic acid (IDS) or an agriculturally acceptable salt thereof, and mixtures thereof.

178. The method of claim 168, wherein the combination further comprises a surfactant.

179. The method of claim 178, wherein the surfactant is selected from the group consisting of an ethoxylated alcohol, a polymeric surfactant, a fatty acid ester, a polyethylene glycol, an ethoxylated alkyl alcohol, a monoglyceride, an alkyl monoglyceride and a mixture thereof.

180. The method of claim 168, wherein the combination further comprises an oil selected from the group consisting of a mineral oil, a vegetable oil and a mixture thereof.

181. The method of claim 180, wherein the oil comprises a mineral oil selected from the group consisting of a paraffinic oil, a branched paraffinic oil, naphthenic oil, an aromatic oil and mixtures thereof.

182. The method of claim 168, wherein the nitrogen-bearing macrocyclic compound and the chelating agent are applied simultaneously to the plant.

183. The method of claim 168, wherein the nitrogen-bearing macrocyclic compound and the chelating agent are applied sequentially to the plant.

184. The method of claim 168, wherein applying the combination to the plant comprises applying a composition comprising the components of the combination, to the plant.

185. The method of claim 168, wherein the combination is applied to the plant by at least one of soil drenching, pipetting, irrigating, spraying, misting, sprinkling, and pouring.

186. The method of claim 168, wherein the microbial pathogen comprises at least one of a fungal pathogen and a bacterial pathogen.

187. The method of claim 168, wherein the plant is a non-woody crop plant, a woody plant or a turfgrass.

188. A composition for inhibiting growth of a microbial pathogen of a plant, comprising: a nitrogen-bearing macrocyclic compound which is a singlet oxygen photosensitizer selected from the group consisting of a porphyrin, a reduced porphyrin and a mixture thereof; a chelating agent to increase permeability of the microbial pathogen to the nitrogen-bearing macrocyclic compound; and a carrier fluid, wherein upon applying the composition to the plant and exposing the plant to light, the nitrogen-bearing macrocyclic compound is activated and generates reactive singlet oxygen species.

Description

DETAILED DESCRIPTION

[0020] Some microbial pathogens, such as Gram-negative bacteria and certain types of fungi have a cellular membrane that is difficult to penetrate. More specifically, these microbial pathogens sometimes have an impermeable outer cell membrane that contains endotoxins and can block small molecules such as antibiotics, dyes and detergents, thereby protecting the sensitive inner membrane and cell wall. It can therefore be challenging to use photodynamic therapy to inhibit growth of certain microbial pathogens in plants because the photosensitizer compounds tend to not achieve good penetration inside the cell wall.

[0021] Photodynamic inhibition of microbial pathogens that are present on plants can be achieved by applying a photosensitizer compound and an enhancer compound. The photosensitizer compound reacts to light by generating reactive oxygen species (ROS), while the enhancer compound increases the overall impact of suppression of the growth of the microbial pathogens, for example by increasing the permeability of the outer membrane of the microbial pathogens to the photosensitizer compound.

[0022] In an implementation, the photosensitizer compound is a porphyrin or a reduced porphyrin compound, such as a chlorin compound, and the enhancer is a chelating agent. An exemplary porphyrin compound is Mg-chlorophyllin, an exemplary chlorin compound is chlorin e6, and an exemplary chelating agent is ethylenediaminetetraacetic acid (EDTA) or agriculturally acceptable salts thereof.

[0023] In some scenarios, the combined use of a photosensitizer compound and a chelating agent have been found to provide enhanced suppression of microbial pathogen growth after photodynamic treatment compared to each used individually. More details regarding the photosensitizer compounds, chelating agents and other additives are provided in the present description.

[0024] It should be understood that when a combination of photosensitizer compound, a chelating agent and any other optional other additives or adjuvants is described throughout the present description and claims, an agriculturally effective amount of each one of the components of the combination can be used so as to provide the anti-microbial activity while being minimally or non-phytotoxic to the host plant.

Photosensitizer Compounds

[0025] As discussed above, photosensitizer compounds can be used to enable photodynamic inhibition of microbial pathogens that are present on plants. The photosensitizer compounds react to light by generating reactive oxygen species (ROS).

[0026] Depending on the type of ROS generated, photosensitizers can be classified into two classes, namely Type I photosensitizers and Type II photosensitizers. On the one hand, Type I photosensitizers form short lived free radicals through electron abstraction or transfer from a substrate when excited at an appropriate wavelength in the presence of oxygen. On the other hand, Type II photosensitizers form a highly reactive oxygen state known as singlet oxygen, also referred to herein as reactive singlet oxygen species. Singlet oxygens are generally relatively long lived and can have a large radius of action.

[0027] It should be understood that the photosensitizer compound can be metallated or non-metallated. When metallated, as can be the case for various nitrogen-bearing macrocyclic compounds that are complexed with a metal, the metal can be selected based on the corresponding ROS type and availability (Type I or Type II) in response to light exposure. For example, when Chlorin photosensitizer compounds are metallated with copper, the ROS that are generated (Type I) tend to have low availability for microbial inhibition, for instance due to a very short half-life. In contrast, when the same photosensitizer compounds are metallated with other metals, such as magnesium, the ROS that are generated have higher availability for microbial inhibition. Thus, when metallated photosensitizer compounds are used, the metal can be selected to obtain a Type II photosensitizer and thereby provide enhanced ROS availability that can in turn facilitate suppression of microbial growth.

[0028] It should be understood that the term singlet oxygen photosensitizer, as used herein, refers to a compound that produces reactive singlet oxygen species when excited by light. In other words, the term refers to a photosensitizer in which the Type II process defined above is dominant compared to the Type I process.

[0029] In some implementations, the photosensitizer compound is a photosensitive nitrogen-bearing macrocyclic compound that can include four nitrogen-bearing heterocyclic rings linked together. In some implementations, the nitrogen-bearing heterocyclic rings are selected from the group consisting of pyrroles and pyrrolines, and are linked together by methine groups (i.e., CH-groups) to form tetrapyrroles. The nitrogen-bearing macrocyclic compound can for example include a porphyrin compound (four pyrrole groups linked together by methine groups), a chlorin compound (three pyrrole groups and one pyrroline group linked together by methine groups), a bacteriochlorin compound or an isobacteriochlorin compound (two pyrrole groups and two pyrroline groups linked together by methine groups), or porphyrinoids (such as texaphrins or subporphyrins), or a functional equivalent thereof having a heterocyclic aromatic ring core or a partially aromatic ring core (i.e., a ring core which is not aromatic through the entire circumference of the ring), or again multi-pyrrole compounds (such as boron-dipyrromethene). It should also be understood that the term nitrogen-bearing macrocyclic compound can be one of the compounds listed herein, or can be a combination of the compounds listed herein. The nitrogen-bearing macrocyclic compound can therefore include a porphyrin, a reduced porphyin, or a mixture thereof. Such nitrogen-bearing macrocyclic compounds can also be referred to as multi-pyrrole macrocyclic compounds (e.g., tetra-pyrrole macrocyclic compounds).

[0030] It should be understood that the term reduced porphyrin as used herein, refers to the group consisting of chlorin, bacteriochlorin, isobacteriochlorin and other types of reduced porphyrins such as corrin and corphin. It should be understood that the nitrogen-bearing macrocyclic compound can be a metal macrocyclic complex (e.g., a Mg-porphyrin) or a non-metal macrocycle (e.g., chlorin E6, Protoporphyrin IX or Tetra PhenylPorphyrin). The nitrogen-bearing macrocyclic compound can be an extracted naturally-occurring compound, or a synthetic compound.

[0031] In implementations where the porphyrin or the reduced porphyrin compound is metallated, the metal can be chosen such that the metallated nitrogen-bearing macrocyclic compound is a Type II photosensitizer (or a singlet oxygen photosensitizer) that generates reactive singlet oxygen species. For, example in the case of chlorins, non-limiting examples of metals that can enable generation of reactive singlet oxygen species through the formation of a Type II photosensitizer are Mg, Zn, Pd, Al, Pt, Sn or Si.

[0032] It should be understood that selecting metals that do not allow for the formation of Type II photosensitizers typically results in a much lower inhibition of the growth of microbial pathogens, at least because no or less reactive singlet oxygen species are generated. Non-limiting examples of metals that are known to not form Type II photosensitizers when complexed with chlorins are Cu, Co, Fe, Ni and Mn.

[0033] It should also be understood that the specific metals that can lead to the formation of Type II photosensitizers versus metals that do not allow for the formation of Type II photosensitizers may vary depending on the type of nitrogen-bearing macrocyclic compound to which it is to be bound. It should also be understood that non-metallated nitrogen-bearing macrocyclic compounds can be Type II photosensitizers. For example, chlorin e6 is a Type II photosensitizer.

[0034] It should be understood that the nitrogen-bearing macrocyclic compound to be used in the methods and compositions of the present description can also be selected based on their toxicity to humans or based on their impact on the environment. For example, porphyrins and reduced porphyrins tend to have a lower toxicity to humans as well as enhanced environmental biodegradability properties when compared to other types of nitrogen-bearing macrocyclic compounds such as phthalocyanines.

[0035] The following formulae illustrate several example nitrogen-bearing macrocyclic compounds described herein:

##STR00001## ##STR00002##

[0036] The nitrogen-bearing macrocyclic compounds such as Zn-TPP and Mg-Chlorophyllin can be obtained from various chemical suppliers such as Organic Herb Inc., Sigma Aldrich or Frontier Scientific. In some scenarios, the nitrogen-bearing macrocyclic compounds are not 100% pure and may include other components such as organic acids and carotenes. In other scenarios, the nitrogen-bearing macrocyclic compounds can have a high level of purity.

[0037] In some implementations, the photosensitizer compound can include multi-pyrrole linear compounds such as bilirubin, boron dipyrrimethene or similar compounds. In some implementations, the photosensitizer compound can include other types of compounds (linear or macrocyclic). A non-limiting example of photosensitizer compound includes diarylheptanoid compounds such as curcumin.

Enhancer Compounds

[0038] The enhancer compound, also referred to herein as a permeabilizing compound, can increase the overall impact of the photosensitizer compound on the inhibition of growth of the microbial pathogens. For example, the enhancer compound can increase the permeability of the outer membrane of the microbial pathogens to the photosensitizer compound.

[0039] In some implementations, the enhancer compound or permeabilizing compound includes a chelating agent. It should be understood that the term chelating agent, as used herein, refers generally to a compound that can form several bonds to a single metal or ion.

[0040] In some implementations, the chelating agent can include at least one carboxylic group, at least one hydroxyl group, at least one phenol group and/or at least one amino group or an agriculturally acceptable salt thereof. In some implementations, the chelating agent can include an aminocarboxylic acid compound or an agriculturally acceptable salt thereof. The aminocarboxylic acid or agriculturally acceptable salt thereof can include an amino polycarboxylic acid or an agriculturally acceptable salt thereof. For example, the amino polycarboxylic acid can include two amino groups and two alkylcarboxyl groups bound to each amino group. The alkylcarboxyl groups can be methylcarboxyl groups.

[0041] In some implementations, the chelating agent is selected from the group consisting of: an aminopolycarboxylic acid, an aromatic or aliphatic carboxylic acid, an amino acid, a phosphonic acid, and a hydroxycarboxylic acid or an agriculturally acceptable salt thereof.

[0042] In some implementations, the methods and compositions described herein include one or more aminopolycarboxylic acid chelating agents. Examples of aminopolycarboxylic acid chelating agents include, without limitation, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTP A), hydroxyethylenediaminetriacetic acid (HEDTA), and ethylenediaminedisuccinate (EDDS), cyclohexanediaminetetraacetic acid (CDTA), N-(2-hydroxyethyl)ethylenediaminetriacetic acid (EDTA-OH) glycol ether diaminetetraacetic acid (GEDTA), alanine diacetic acid (ADA), alkoyl ethylene diamine triacetic acids (e.g., lauroyl ethylene diamine triacetic acids (LED3 A)), asparticaciddiacetic acid (ASDA), asparticacidmonoacetic acid, diamino cyclohexane tetraacetic acid (CDTA), 1,2-diaminopropanetetraacetic acid (DPTA-OH), 1,3-diamino-2-propanoltetraacetic acid (DTP A), diethylene triamine pentam ethylene phosphonic acid (DTPMP), diglycolic acid, dipicolinic acid (DP A), ethanolaminediacetic acid, ethanoldiglycine (EDG), ethylenediaminediglutaric acid (EDDG), ethylenediaminedi(hydroxyphenylacetic acid (EDDHA), ethylenediaminedipropionic acid (EDDP), ethylenediaminedisuccinate (EDDS), ethylenediaminemonosuccinic acid (EDMS), ethylenediaminetetraacetic acid (EDTA), ethylenediaminetetrapropionic acid (EDTP), and ethyleneglycolaminoethylestertetraacetic acid (EGTA) and agriculturally acceptable salts (for example, the sodium salts, calcium salts and/or potassium salts) thereof.

[0043] One non-limiting example of chelating agent is ethylenediaminetetraacetic acid (EDTA) or an agriculturally acceptable salt thereof. The aminocarboxylate salt can for example be a sodium or calcium salt. EDTA can be represented as follows:

##STR00003##

[0044] Another non-limiting example of chelating agent is polyaspartic acid or an agriculturally acceptable salt thereof (i.e., a polyaspartate), such as sodium polyaspartate, which can be generally represented as follows. The molecular weight of the polyaspartate salt can for example be between 2,000 and 3,000.

##STR00004##

[0045] The chelating agent can thus be a polymeric compound, which can include aspartate units, carboxylic groups, and other features found in polyaspartates. The polyaspartate can be a co-polymer that has alpha and beta linkages, which may be in various proportions (e.g., 30% alpha, 70% beta, randomly distributed along the polymer chain). One non-limiting example of a sodium polyaspartate is Baypure DS 100, which can be represented as follows.

##STR00005##

[0046] Other non-limiting examples of chelating agents include EDDS (ethylenediamine-N,N-disuccinic acid), IDS (iminodisuccinic acid (N-1,2-dicarboxyethyl)-D,L-aspartic acid), isopropylamine, triethanolamine, triethylamine, ammonium hydroxide, tetrabutylammonium hydroxide, hexamine, GLDA (L-glutamic acid N,N-diacetic acid), or agriculturally acceptable salts thereof. The chelating agent can be metallated or non-metallated.

[0047] IDS can be used as a tetrasodium salt of IDS (e.g., tetrasodium iminodisuccinate), which can be Baypure CX100, represented as follows:

##STR00006##

[0048] EDDS can be used as a trisodium salt of EDDS, represented as follows:

##STR00007##

[0049] GLDA can be used as a tetrasodium salt of GLDA, represented as follows:

##STR00008##

[0050] In some implementations, the methods and compositions described herein include one or more amino acid chelating agents. Examples of amino acid chelating agents include, without limitation, alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tyrosine, valine, and salts (for example, the sodium salts, calcium salts and/or potassium salts) and combinations thereof.

[0051] In some implementations, the methods and compositions described herein include one or more aromatic or aliphatic carboxylic acid chelating agents. Examples of aromatic or aliphatic carboxylic acid chelating agents include, without limitation, oxalic acid, succinic acid, pyruvic acid malic, acid, malonic acid, salicylic acid, and anthranilic acid, and salts (for example, the sodium salts, calcium salts and/or potassium salts) thereof. In some implementations, the methods and compositions described herein include one or more polyphenol chelating agents. One non-limiting examples of a polyphenol chelating agent is tannins such as tannic acid.

[0052] In some implementations, the methods and combinations described herein include one or more hydroxycarboxylic acid chelating agents. Examples of the hydroxycarboxylic acid type chelating agents include, without limitation, malic acid, citric acid, glycolic acid, heptonic acid, tartaric acid and salts (for example, the sodium salts, calcium salts and/or potassium salts) thereof.

[0053] In some implementations, the one or more chelating agents can be applied as the free acid, as an agriculturally acceptable salt, or combinations thereof.

[0054] In some implementations, each of one or more the chelating agent(s) is applied as the free acid. In other implementations, the chelating agent(s) can be applied as a salt. Exemplary salts include sodium salts, potassium salts, calcium salts, ammonium salts, amine salts, amide salts, and combinations thereof. In still other implementations, when more than one chelating agent is present, at least one of the chelating agents is applied as a free acid, and at least one of chelating agents is applied as a salt.

[0055] When the components are provided as part of a single composition, the composition can be provided to have certain concentrations and relative proportions of components. For example, the composition can have between about 100 nM and about 50 mM, between about 5 micromolar and about 10 mM, between about 1 micromolar and about 1000 micromolar, between about 5 micromolar and about 200 micromolar of the nitrogen-bearing macrocyclic compound, between about 10 micromolar and about 150 micromolar of the nitrogen-bearing macrocyclic compound, between about 25 micromolar and about 100 micromolar of the nitrogen-bearing macrocyclic compound, or between about 50 micromolar and about 75 micromolar of the nitrogen-bearing macrocyclic compound.

[0056] The composition can also have between about 2 micromolar and about 10,000 micromolar of the chelating agent, between about 5 micromolar and about 5,000 micromolar of the chelating agent, between about 10 micromolar and about 1,000 micromolar of the chelating agent, between about 25 micromolar and about 500 micromolar of the chelating agent, between about 50 micromolar and about 100 micromolar of the chelating agent, for example.

[0057] The relative proportion, by weight, of the nitrogen-bearing macrocyclic compound and the chelating agent in the composition can be between about 50:1 and about 1:1000, between about 20:1 and about 1:500, between about 10:1 and about 1:100, or between about 1:1 and about 1:10, for example.

Additives and Adjuvants

[0058] In some implementations, the methods and compositions described herein include one or more agriculturally suitable adjuvants.

[0059] In some implementations, 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) and one or more utility adjuvants (e.g., 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 conditioners; one or more buffers; one or more anti-foaming agents; one or more UV blockers; one or more antioxidants; one or more fertilizers, nutrients, and/or micronutrients; and/or one or more herbicide safeners). Exemplary adjuvants are provided in Hazen, J. L. Weed Technology 14: 773-784 (2000), which is incorporated by reference in its entirety.

[0060] In some implementations, oil can be combined with the nitrogen-bearing macrocyclic compound. The oil can be selected from the group consisting of a mineral oil, a vegetable oil and a mixture thereof.

[0061] Non-limiting examples of vegetable oils include oils that include medium chain triglycerides (MCT), oil extracted from nuts. Other non-limiting examples of vegetable oils include coconut oil, canola oil, soybean oil, rapeseed oil, sunflower oil, safflower oil, peanut oil, cottonseed oil, palm oil, rice bran oil or mixtures thereof. Non-limiting examples of mineral oils include paraffinic oils, branched paraffinic oils, naphthenic oils, aromatic oils or mixtures thereof.

[0062] Non-limiting examples of paraffinic oils include various grades of poly-alpha-olefin (PAO). For example, the paraffinic oil can include HT60, HT100, High Flash Jet, LSRD, and N65DW. The paraffinic oil can include a paraffin having a number of carbon atoms ranging from about 12 to about 50, or from about 16 to 35. In some scenarios, the paraffin can have an average number of carbon atoms of 23. In some implementations, the oil can have a paraffin content of at least 80 wt %, or at least 90 wt %, or at least 99 wt %.

[0063] The nitrogen-bearing macrocyclic compound and the oil can be applied in a relative proportion, by weight, between about 50:1 and about 1:1000, between about 20:1 and about 1:500, between about 10:1 and about 1:100, or between about 1:1 and about 1:10, for example.

[0064] The nitrogen-bearing macrocyclic compound and the oil can be added sequentially or simultaneously. When added simultaneously, the nitrogen-bearing macrocyclic compound and the oil can be added as part of the same composition or as part of two separate compositions. In some implementations, the nitrogen-bearing macrocyclic compound and the oil can be combined in an oil-in-water emulsion. That is, the combination can include the nitrogen-bearing macrocyclic compound combined with the oil and water so that the combination is formulated as an oil-in-water emulsion. The oil-in-water emulsion can also include other additives such as a chelating agent, a surfactant or combinations thereof.

[0065] As used herein, the term oil-in-water emulsion refers to a mixture in which one of the oil (e.g., the paraffinic oil) and water is dispersed as droplets in the other (e.g., the water). In some implementations, an oil-in-water emulsion is prepared by a process that includes combining the paraffinic oil, water, and any other components and the paraffinic oil and applying shear until the emulsion is obtained. In other implementations, an oil-in-water emulsion is prepared by a process that includes combining the paraffinic oil, water, and any other components in the mixing tank and spraying through the nozzle of a spray gun.

[0066] In some implementations, the nitrogen-bearing macrocyclic compound and the chelating agent are part of a composition that includes a carrier fluid. A suitable carrier fluid allows obtaining a stable solution, suspension and/or emulsion of the components of the composition in the carrier fluid. In some implementations, the carrier fluid is water. In other implementations, the carrier fluid is a mixture of water and other solvents or oils that are non-miscible or only partially soluble in water.

[0067] It should also be understood that the compositions and combinations of the present description can be provided separately or together in the same composition. In some implementations, the components of the compositions of the present description can be packaged in a concentrated form, without the carrier fluid, and the carrier fluid (e.g., water) can be added to form directly by the operator that applies the composition to plants in order to form the composition to be applied.

[0068] In some implementations, a combination of nitrogen-bearing macrocyclic compound and oil can be used to inhibit growth of a microbial pathogen in a plant. The combination can be an oil-in-water emulsion, where the surfactant is selected such that the nitrogen-bearing macrocyclic compound is maintained in dispersion in the oil-in-water emulsion for delivery to the plant.

[0069] The combination can include a surfactant (also referred to as an emulsifier). The surfactant can be selected from the group consisting of an ethoxylated alcohol, a polymeric surfactant, a fatty acid ester, a polyethylene glycol, an ethoxylated alkyl alcohol, a monoglyceride, an alkyl monoglyceride, an amphipathic glycoside, and a mixture thereof. For example, the fatty acid ester can be a sorbitan fatty acid ester. The surfactant can include a plant derived glycoside such as a saponin. The surfactant can be present as an adjuvant to aid coverage of plant foliage. The surfactant can be an acceptable polysorbate type surfactant (e.g., Tween 80), a nonionic surfactant blend (e.g., Altox 3273), or another suitable surfactant. In some implementations, the polyethylene glycol can include a polyethylene glycol of Formula:

R.sup.1O(CH.sub.2CH.sub.2O).sub.fR.sup.2

wherein R.sup.1H, CH.sub.2CHCH.sub.2 or COCH.sub.3; R.sup.2H, CH.sub.2CHCH.sub.2 or COCH.sub.3; and f1.

Combination of Photosensitizer and Enhancer Compounds

[0070] The photosensitizer compound and the enhancer compound can be provided as part of an anti-microbial composition. The anti-microbial composition can also include a delivery fluid, such as water, as well as other additives.

[0071] The anti-microbial composition can be provided to have certain concentrations and relative proportions of components. For example, the anti-microbial composition can 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 of the photosensitizer compound, between about 25 micromolar and about 100 micromolar of the photosensitizer compound, or between about 50 micromolar and about 75 micromolar of the photosensitizer compound.

[0072] The anti-microbial composition can also have between about 2 micromolar and about 10,000 micromolar of the enhancer compound, between about 5 micromolar and about 5,000 micromolar of the enhancer compound, between about 10 micromolar and about 1,000 micromolar of the enhancer compound, between about 25 micromolar and about 500 micromolar of the enhancer compound, between about 50 micromolar and about 100 micromolar of the enhancer compound, for example.

[0073] The relative proportion, by weight, of the photosensitizer compound and the enhancer compound in the anti-microbial composition can be between about 50:1 and about 1:1000, between about 20:1 and about 1:500, between about 10:1 and about 1:100, or between about 1:1 and about 1:10, for example.

[0074] In terms of other additives that can be present in the anti-microbial compositions, a surfactant can be present as an adjuvant to aid coverage of plant foliage. The surfactant can be an acceptable polysorbate type surfactant (e.g., Tween 80), a nonionic surfactant blend (e.g., Altox 3273), or another suitable surfactant.

Application of Photosensitizer and Enhancer Compounds

[0075] The photosensitizer compound and the enhancer compound can be applied to plants for photodynamic inhibition of microbial pathogens. The photosensitizer compound and the enhancer compound can be applied simultaneously to the plants. For example, an anti-microbial composition can be prepared to include the photosensitizer and enhancer compounds as well as a delivery fluid, such as water or a water-oil emulsion for example. The anti-microbial composition can be applied to the plant by spraying, misting, sprinkling, pouring, or any other suitable method. The anti-microbial composition can be applied to the foliage, roots and/or stem of the plant. Other additives can also be included in the anti-microbial composition, and other application methods can also be performed.

[0076] The plants on which the anti-microbial composition is applied can be outdoors or indoors (e.g., greenhouse) where they are exposed to natural sunlight, or in an indoor location where they are exposed to artificial light. The exposure to the incident light is provided such that the photosensitizer compound can generate ROS that, in turn, facilitate disruption of microbial growth.

[0077] In operation, the photosensitizer compound and the enhancer compound are brought into contact with the microbial pathogen that has infected a plant. The photosensitizer compound and the enhancer compound both come into contact with the cell walls and intercellular material of the pathogenic microbes.

[0078] Various mechanisms of action can be facilitated by the combination of the photosensitizer and enhancer compounds. For example, the enhancer compound can interact with the photosensitizer compound and/or with species present at the cell walls of the microbial pathogens, to disrupt the cell walls or enhance access or penetration of the photosensitizer compound, such that the phototreatment and consequent ROS generation can have an increased inhibitory impact on the microbial pathogens. The interactions of the enhancer compound can depend on the structures of the photosensitizer compound and the enhancer compound. For example, chelating agents such as EDTA can complex with metals that are present within the macrocycle of certain photosensitizer compounds and/or with counter-ions that are present at the cell walls of the microbial pathogens.

Microbial Pathogens and Plants

[0079] The microbial pathogens to which the anti-microbial composition can be applied include fungal and bacterial pathogens.

[0080] The fungal pathogens to which the anti-microbial composition can be applied include Alternaria solani, which can infect plants such as tomatoes and potatoes; Botrytis cinerea, which can infect grapes, as well as soft fruits and bulb crops; or Sclerotinia homoeocarpa, which can commonly infect turfgrasses. Other fungal pathogens in the Alternaria, Botrytis or Sclerotinia genera can also receive application of the anti-microbial composition. The anti-microbial composition can be applied to plants that are affected or susceptible to pathogens that cause various plant diseases, e.g., 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., or other fungal pathogens.

[0081] The bacterial pathogens to which the anti-microbial composition can be applied include gram-negative bacteria, such as Erwinia amylovara, or other bacterial pathogens in the genus Erwinia that can infect woody plants. E. amylovara causes fire blight on various plants, including pears, apples, and other Rosaceae crops. The anti-microbial composition can be applied to plants that are affected or susceptible to pathogens that cause various plant diseases, e.g., Pseudomonas, Xanthomonas, Agrobacterium, Curtobacterium, Streptomyces, E. Coli, Xylella fastidiosa (which causes Olive Quick Decline Syndrome (OQDS) disease), or other bacterial pathogens.

[0082] The anti-microbial composition can be used for various types of plants that are affected by microbial pathogens. Crop plants, lawn plants, trees and other plants infected with microbial pathogens can be treated.

[0083] It is also noted that the anti-microbial compositions described herein can have various inhibitory effects on the microbial pathogens depending on the type of plant and pathogen as well as the state of microbial infection. While herein it is described that the anti-microbial composition can inhibit microbial pathogen growth on a plant, such expressions should not be limiting but should be understood to include suppression of microbial pathogens, prevention against microbial pathogens, destruction of microbial pathogens or generally increasing toxicity toward microbial pathogens.

Types of Plants

[0084] The compound or composition may be used for various types of plants that may be affected microbial pathogens. The plant can be a non-woody crop plant, a woody plant or a turfgrass. The plant can be selected from the group consisting of a crop plant, a fruit plant, a vegetable plant, a legume plant, a cereal plant, a fodder plant, an oil seed plant, a field plant, a garden plant, a green-house plant, a house plant, a flower plant, a lawn plant, a turfgrass, a tree such as a fruit-bearing tree, and other plants that may be affected by microbial pathogens.

[0085] In some implementations, the plant is a turfgrass. As used herein, the term turfgrass refers to a cultivated grass that provides groundcover, for example a turf or lawn that is periodically cut or mowed to maintain a consistent height. Grasses belong to the Poaceae family, which is subdivided into six subfamilies, three of which include common turfgrasses: the Festucoideae subfamily of cool-season turfgrasses; and the Panicoideae and Eragrostoideae subfamiles of warm-season turfgrasses. A limited number of species are in widespread use as turfgrasses, generally meeting the criteria of forming uniform soil coverage and tolerating mowing and traffic. In general, turfgrasses have a compressed crown that facilitates mowing without cutting off the growing point. In the present context, the term turfgrass includes areas in which one or more grass species are cultivated to form relatively uniform soil coverage, including blends that are a combination of differing cultivars of the same species, or mixtures that are a combination of differing species and/or cultivars.

Synergistic Effect of the Combinations

[0086] In some scenarios, the combinations can exhibit a synergistic response for inhibiting growth of microbial pathogens in plants. It should be understood that the terms synergy or synergistic, as used herein, refer to the interaction of two or more components of a combination (or composition) so that their combined effect is greater than the sum of their individual effects, this may include, in the context of the present description, the action of two or more of the nitrogen-bearing macrocyclic compound, the oil and the chelating agent. In some scenarios, the nitrogen-bearing macrocyclic compound and the oil can be present in synergistically effective amounts. In some scenarios, the nitrogen-bearing macrocyclic compound and the chelating agent can be present in synergistically effective amounts. In some scenarios, the oil and the chelating agent can be present in synergistically effective amounts. In some scenarios, the nitrogen-bearing macrocyclic compound, the oil and the chelating agent can be present in synergistically effective amounts.

[0087] In some scenarios, the approach as set out in S. R. Colby, Calculating synergistic and antagonistic responses of herbicide combinations, Weeds 15, 20-22 (1967), can be used to evaluate synergy. Expected efficacy, E, may be expressed as: E=X+Y(100X)/100, where X is the efficacy, expressed in % of the untreated control, of a first component of a combination, and Y is the efficacy, expressed in % of the untreated control, of a second component of the combination. The two components are said to be present in synergistically effective amounts when the observed efficacy is higher than the expected efficacy.

EXAMPLES & EXPERIMENTATION

[0088] Throughout the Examples, it should be understood that the % values provided in compositions refer to wt % values. Furthermore, the balance to 100 wt % of the listed % values is always a corresponding amount of water. For example: the entry 0.1% MgChl+0.1% EDTA-Ca+0.04% Atlox3273 means a composition consisting of 0.1 wt % MgChl, 0.1 wt % EDTA-Ca, 0.04 wt % Atlox3273 and water to arrive at 100 wt %.

Example 1

[0089] In this example, application of a porphyrin photosensitizer compound and a chelating agent suppresses growth of Bacteria (Erwinia amylovora) that causes Fire Blight disease using photodynamic inhibition. In particular, Mg-chlorophyllin and disodium EDTA are used and their combination shows to inactivate E. amylovora growth.

[0090] E. amylovora is grown in liquid medium. Samples are incubated for 30 min with 100 M of Mg-Chlorophyllin with or without 5 mM of Disodium-EDTA, and then illuminated with 395 nm (fluence 26.6 J cm.sup.2) for 30 mins. CFU of the bacteria is counted on the plates. The following table summarizes the results:

TABLE-US-00001 TABLE 1 Results on relative inactivation of bacteria after PDI treatment Treatment Relative inactivation, CFU untreated control-dark 1 untreated control-light 0.9 Mg-Chlorophyllin (100 M) without EDTA 1.9 (5 mM) in light Mg-Chlorophyllin (100 M) with EDTA 2.1 10.sup.4 (5 mM) in light EDTA (5 mM) alone in light 11.1

[0091] The phototreatment of chlorophyllin or EDTA alone has little or no effect on E. amylovora inactivation at the tested concentration. Combining EDTA with chlorophyllin increases the antibacterial effect by about 1000-fold at the tested concentration. E. amylovora is almost completely inactivated with the combination of the two compounds under the test conditions.

Example 2

[0092] In this example, application of a porphyrin photosensitizer compound and a chelating agent suppressed growth of fungal pathogen Alternaria solani using photodynamic inhibition. In particular, Mg-chlorophyllin and disodium EDTA were used and their combination was shown to inactivate Alternaria solani growth.

[0093] In the experiments, Alternaria solani mycelia were grown in liquid medium for 24 hours. Small spheres of the mycelia (average diameter 2 mm) were incubated for 100 minutes with 100 M of Mg-Chlorophyllin with 0 to 5 mM of Disodium-EDTA. Samples were illuminated with 395 nm (fluence 106.6 J cm.sup.2) for 120 mins and the radial growth of mycelial patches after 7 days on agar medium was measured. A sample was considered dead, if there was no growth observable after 7 days on agar plates. The following table summarizes the results:

TABLE-US-00002 TABLE 2 Results on percentage of dead Alternaria solani mycelia Treatment dead % Mg-Chlorophyllin (100 M) without EDTA (0M) in light 11.5% Mg-Chlorophyllin (100 M) with EDTA (5 M) in light 33.3% Mg-Chlorophyllin (100 M) with EDTA (50 M) in light 66.7% Mg-Chlorophyllin (100 M) with EDTA (500 M) in light 83.3% Mg-Chlorophyllin (100 M) with EDTA (5 mM) in light 94.1% EDTA (5 mM) alone in light 0% EDTA (50 mM) alone in light 0% EDTA (100 mM) alone in light 0% EDTA (500 mM) alone in light 0% EDTA (5 mM) alone no light 0% EDTA (50 mM) alone no light 0% EDTA (100 mM) alone no light 11.1% EDTA (500 mM) alone no light 11.1%

[0094] The phototreatment of chlorophyllin or EDTA alone had little or no effect on fungal mycelia. Adding a combination of EDTA and 100 m Chlorophyllin increased the suppression on the growth of fungal mycelia. Alternaria solani was almost completely inactivated with the combination of 100 M Chlorophyllin and 5 mM disodium-EDTA.

Example 3

[0095] In this example, application of a porphyrin photosensitizer compound and a chelating agent suppressed growth of Botrytis cinerea fungi that causes Gray Mold disease using photodynamic inhibition. In particular, Mg-chlorophyllin and disodium EDTA were used and their combination was shown to inactivate Botrytis cinerea growth.

[0096] In the experiments, Botrytis cinerea mycelia were grown in liquid medium for 48 hours. Small spheres of the mycelia (average diameter 2 mm) were incubated for 100 minutes with Mg-Chlorophyllin with or without 5 mM Disodium-EDTA. Samples were illuminated with 395 nm (fluence 106.6 J cm.sup.2) for 120 minutes and the radial growth of mycelial patches after 7 days on agar medium was measured. A sample was considered dead, if there was no growth observable after 7 days on agar plates. The following table summarizes the results:

TABLE-US-00003 TABLE 3 Results on percentage of dead B. cinerea mycelia Treatment dead % Untreated control in dark 0 Untreated control with light 0 EDTA alone in dark (5 mM) 0 EDTA alone with light (5 mM) 0 Mg-Chlorophyllin (1 M) with EDTA (5 mM) in light 0 Mg-Chlorophyllin (10 M) with EDTA (5 mM) in light 33.3% Mg-Chlorophyllin (100 M) with EDTA (5 mM) in light 91.7% Mg-Chlorophyllin (10 M) alone in light 0 Mg-Chlorophyllin (100 M) alone in light 0

[0097] The phototreatment of chlorophyllin or EDTA alone had little or no effect on fungal mycelia. Adding EDTA into Chlorophyllin increased the suppression on the growth of fungal mycelia, notably at Chlorophyllin concentrations of 10 M or above at the test conditions. Botrytis cinerea was largely inactivatd when treated by combination of 100 M Chlorophyllin and 5 mM disodium-EDTA.

Example 4

[0098] In this example, application of a porphyrin photosensitizer compound and a chelating agent suppressed growth of Botrytis cinerea fungi that causes Gray Mold disease using photodynamic inhibition. In particular, Mg-chlorophyllin and calcium disodium EDTA or disodium EDTA were used, and the combination of the porphyrin photosensitizer compound and each chelating agent was shown to inactivate Botrytis cinerea growth.

[0099] In the experiments, Botrytis cinerea mycelia were grown in liquid medium for 48 hours. Small spheres of the mycelia (average diameter 2 mm) were incubated for 100 minutes with Mg-Chlorophyllin with or without 5 mM EDTA (calcium or disodium). Samples were illuminated with 395 nm (fluence 106.6 J cm.sup.2) for 120 min and the radial growth of mycelial patches after 7 days on agar medium was measured. A sample was considered dead, if there was no growth observable after 7 days on agar plates. The following table summarizes the results:

TABLE-US-00004 TABLE 4 Results on percentage of dead B. cinerea mycelia for Na or Ca EDTA dead % Treatment-with Ca-EDTA Untreated control in dark 0 Untreated control with light 0 EDTA-Ca alone with light (5 mM) 0 Mg-Chlorophyllin (100 M) with EDTA-Ca (5 mM) in light 47.6% Mg-Chlorophyllin (100 M) alone in light 0 Treatment-with Na-EDTA Untreated control in dark 0 Untreated control with light 0 EDTA-Na alone with light (5 mM) 0 Mg-Chlorophyllin (100 M) with EDTA-Na (5 mM) in light 91.7% Mg-Chlorophyllin (100 M) alone in light 0

[0100] The phototreatment of chlorophyllin or EDTA alone (including calcium and disodium EDTA) had limited effect on fungal mycelia. For both tested chelating agents, adding EDTA into Chlorophyllin increased the suppression on the growth of fungal mycelia, notably at Chlorophyllin concentrations of 10 uM or above at the test conditions. Botrytis cinerea was greatly inactivated when treated by combination of 100 M Chlorophyllin and 5 mM Na-EDTA, and was also inactivated when treated by combination of 100 M Chlorophyllin and 5 mM Ca-EDTA. The efficiency was lower than with Na-EDTA.

Example 5

[0101] In this example, application of a porphyrin photosensitizer compound and a chelating agent suppressed growth of Early Blight fungi (Alternaria solani) using photodynamic inhibition. In particular, Chlorin E6 and disodium EDTA were used, and the combination of the porphyrin photosensitizer compound and the chelating agent was shown to inactivate Alternaria solani growth.

[0102] In the experiments, Alternaria solani mycelia were grown in liquid medium for 24 hours. Small spheres of the mycelia (average diameter 2 mm) were incubated for 100 minutes with Chlorin E6 with or without 5 mM of disodium-EDTA. Samples were illuminated with 395 nm (fluence 106.6 J cm.sup.2) for 120 mins and the radial growth of mycelial patches after 7 days on agar medium was measured. A sample was considered dead, if there was no growth observable after 7 days on agar plates. The following table summarizes the results:

TABLE-US-00005 TABLE 5 Results on percentage of dead Alternaria solani mycelia Treatment dead % no light, 0 M Chlorin E6 0.0% no light, 100 M Chlorin E6 0.0% light, 0 M Chlorin E6 0.0% light, 1 M Chlorin E6 0.0% light, 10 M Chlorin E6 8.3% light, 100 M Chlorin E6 .sup.0% no light, 5 mM Na-EDTA alone .sup.0% no light, 100 M Chlorin E6 + 5 mM Na-EDTA .sup.0% light, 5 mM Na-EDTA alone .sup.0% light, 1 M Chlorin E6 + 5 mM Na-EDTA .sup.0% light, 10 M Chlorin E6 + 5 mM Na-EDTA 88.8% light, 100 M Chlorin E6 + 5 mM Na-EDTA 16.7%

[0103] The phototreatment of Chlorin E6 or EDTA alone had little or no effect on fungal mycelia. Adding EDTA into Chlorin E6 increased the suppression on the growth of fungal mycelia, notably at Chlorophyllin concentrations of 10 M or above at the test conditions. Alternaria solani was largely inactivated when treated by combination of 10 uM Chlorophyllin and 5 mM disodium-EDTA.

Example 6Test on Host Plant Creeping Bentgrass

[0104] In this example, control of dollar spot (Sclerotinia homoeocarpa) on creeping bentgrass was assessed. In terms of methodology, bentgrass (L-93) were grown in 3.5 inch pots for 4-6 weeks. Plants were inoculated with 0.2 g/pot of wheat seed inoculum containing 5 different isolates of Sclerotinia homoeocarpa (dollar spot pathogen). After 24 hrs, inoculated plants were sprayed with different formula. Four reps of each treatment were done. Following foliar application, plants were incubated in the dark for 8 hr, then placed randomly under the LED lights (PAR ca. 300 uMoles/m.sup.2/sec). Four reps of each treatment were done. LED Light cycle set from 6 pm OFF, 2 am ON, to maintain 16 hour:8 hour, light:dark cycle. Seven days post inoculation (DPI), pots of A. stolonifera were assessed for % yellowing of leaf blades and % mycelial coverage of leaf blades with S. homoeocarpa.

TABLE-US-00006 TABLE 6 Results on inhibition of Sclerotinia homoeocarpa on bentgrass Mean % Yellowing Mean % Mycelium Treatment 7 DPI inhibition % 7 DPI inhibition % 1 0.1% MgChln + 20 5.88% 27.5 24% 0.04% Atlox3273 2 0.1%EDTA-Ca + 26.25 23.53% 37.5 3% 0.04% Atlox3273 3 0.1% MgChl + 13.75 35.29% 20 45% 0.1% EDTA-Ca + 0.04% Atlox3273 Control + non-treated, 21.25 36.25 p inoculated control Control non-treated, non- 0 0 p inoculated control

[0105] Altox3273 is an example of a surfactant that can be used in the composition. Such surfactant compounds can be used as adjuvants to aid coverage of plant foliage, for example.

[0106] From the above results, it can be seen that the combination of MgChl and EDTA provided enhanced photodynamic inhibition on a plant (creeping bentgrass) compared to each compound used alone. In this example, the combination was provided as an aqueous composition that further included a surfactant.

Example 7

[0107] In this example, control of dollar spot fungus (Sclerotinia homoeocarpa) was assessed for Mg Chlorophyllin, sodium polyaspartate, and the combination thereof. Treatments were amended into Potato Dextrose Agar (PDA) at desired concentrations. Then, a 5 mm diameter plug of a Sclerotinia homoeocarpa isolate (3 isolates total tested) was inoculated into the center of the amended Petri-dish and incubated at 21 C. in the dark for 24 hours. After 24 hours, one set of Petri-dishes (in triplicate) was left in the dark and one set was placed under illumination for the remainder of the experiment (all at 21 C.). Radial growth of the fungus was monitored daily until the growth of S. homoeocarpa on non-amended PDA reached the edge of the Petri-dish. Illumination was provided by fluorescent lights emitting about 180 mol/m.sup.2/s photosynthetically active radiation (PAR).

TABLE-US-00007 TABLE 7A Results on inhibition in dark Mean Radial Rate Growth Treatment.sup.1 (M).sup.5 (mm).sup.23 % Inhibition.sup.4 Non-amended 0 13.3 (control) Mg Chlorophyllin (Mg 31 11.2 24.1 Chl) Baypure DS (sodium 50 13.7 7.1 polyaspartate) Baypure DS (sodium 500 10.8 27.1 polyaspartate) Baypure DS (sodium 1000 9 39.1 polyaspartate) Baypure DS (sodium 2000 5.5 62.8 polyaspartate) Mg Chln + Baypure 31 + 50 11.4 22.6 DS Mg Chln + Baypure 31 + 500 10.7 27.4 DS Mg Chln + Baypure 31 + 1000 8.3 44.0 DS Mg Chln + Baypure 31 + 2000 5.4 63.2 DS

TABLE-US-00008 TABLE 7B Results on inhibition in light Mean Radial Rate Growth Treatment.sup.1 (M).sup.5 (mm).sup.2,3 % Inhibition.sup.4 Non-amended (control) 0 13.6 Mg Chlorophyllin 31 6.6 51.0 Baypure DS (sodium 50 14.4 7.0 polyaspartate) Baypure DS (sodium 500 10.7 21.0 polyaspartate) Baypure DS (sodium 1000 7.6 43.6 polyaspartate) Baypure DS (sodium 2000 (1.25%) 4.4 67.5 polyaspartate) Mg Chln + Baypure DS 31 + 50 5.9 56.0 Mg Chln + Baypure DS 31 + 500 5 63.0 Mg Chln + Baypure DS 31 + 1000 3.4 74.9 Mg Chln + Baypure DS 31 + 2000 0.6 95.5 Notes on above Tables: .sup.1Treatments were amended into Potato Dextrose Agar (PDA); .sup.2Means were calculated based on 3 fungal isolates replicated 3 times, with 2 measurements per replicate (18 total measurements); .sup.3Means represent growth that occurred between 24 and 48 hours of incubation at 21 C.; .sup.4% Inhibition calculated relative to non-amended control. .sup.5Mw of Baypure DS listed as 2,000-3,000. Mw of 2,500 g/mol was used for calculations.

[0108] From the above results, it can be seen that the combination of MgChl and polyaspartate provided enhanced photodynamic inhibition compared to each compound used alone.

Example 8

[0109] Xanthomonas axonopodis are bacterial pathogens that cause citrus canker. Citrus canker is a highly contagious plant disease than can destroy an entire crop field. Mg-Chlorophyllin has been found to have little effect to control the bacterial in the assay (same protocol as Example 1, on Erwinia amylovora). However, it has been found that combining Chlorophyllin with EDTA (e.g., 5 mM sodium EDTA) with light illumination provided enhanced control of Xanhomonas axonopodis. Positive results were obtained at several chlorophyllin concentrations and exposure times.

[0110] Xanthomonas were grown in liquid medium. Samples were incubated for 30 mins or 5 mins with Mg-Chlorophyllin and with Disodium-EDTA for 30 mins, and then illuminated with 395 nm (fluence 26.6 J cm2) for 30 mins. CFU of the bacteria was counted on the plates.

TABLE-US-00009 TABLE 8 Results of Xanthomonas Axonopodis count Treatment CFU/mL log CFU/mL Inhibition % Untreated Control in dark 3.04E+07 7.5 Mg-Chlorophyllin 100 M + 2.94E+06 6.5 90.3% 5 mM EDTA, in dark Mg-Chlorophyllin 100 M + 4.09E+02 2.6 100.0% 5 mM EDTA, light 30 min Mg-Chlorophyllin 10 M + 3.92E+03 3.6 100.0% 5 mM EDTA, light 30 min Mg-Chlorophyllin 1 M + 3.62E+05 5.6 98.8% 5 mM EDTA, light 30 min Mg-Chlorophyllin 100 M + 3.33E+02 2.5 100.0% 5 mM EDTA, light 5 min Mg-Chlorophyllin 10 M + 7.32E+04 4.9 99.8% 5 mM EDTA, light 5 min Mg-Chlorophyllin 1 M + 5.69E+05 5.8 98.1% 5 mM EDTA, light 5 min

Example 9 (Curcuminnot a Nitrogen-Bearing Macrocyclic Compound)

[0111] Experiments have indicated that the combination of EDTA and Curcumin provides enhanced photodynamic suppression of fungal growth compared to each component individually. This example indicates that compounds that are linear and/or do not have a macrocyclic core structure, as do porphyrins, can also be combined with chelating agents for photodynamic inhibition applications.

[0112] In this example, control of dollar spot fungus (Sclerotinia homoeocarpa) with Curcumin, EDTA, and the combination was assessed. Curcumin is dissolved in DMSO first and then diluted with 0.25% Tween solution. Treatments were amended into Potato Dextrose Agar (PDA) at desired concentrations. Then, a 5 mm diameter plug of a Sclerotinia homoeocarpa isolate (3 isolates total tested) was inoculated into the center of the amended Petri-dish and incubated at 21 C. in the dark for 24 hours. After 24 hours, one set of Petri-dishes (in triplicate) is left in the dark and on set is placed under illumination for the remainder of the experiment (all at 21 C.). Radial growth of the fungus is monitored daily until the growth of S. homoeocarpa on non-amended PDA reaches the edge of the Petri-dish. Illumination is provided by fluorescent lights emitting about 180 mol/m.sup.2/s photosynthetically active radiation (PAR).

TABLE-US-00010 TABLE 9A Results in dark Mean Radial Rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.4 Non-amended 0 14.2 (control) Curcumin (DMSO) 1000 9.9 33.0 disp. in 0.25% Tween 80 Curcumin (DMSO) 500 13.1 11.4 disp. in 0.25% Tween 80 Curcumin (DMSO), 1000 2.3 84.4 0.25% T80, 100 M Ca-EDTA (RD174) Curcumin (DMSO), 500 3.4 77.0 0.25% T80, 100 M Ca-EDTA (RD174) Tween 80 0.25% 0 14.1 4.89 Ca-EDTA (RD174) 0 2.8 80.8 100 M Tween 80 0.25% + 0 3.06 79.3 Ca-EDTA (RD174) 100 M DMSO (in de-ionized 0 13.5 8.6 water) 0.0736%

TABLE-US-00011 TABLE 9B Results on inhibition in light Mean Radial Curcumin rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.4 Non-amended (control) 0 12.7 Curcurmin (DMSO) disp. 1000 9.6 28.9 in 0.25% Tween 80 Curcurmin (DMSO) disp. 500 12.4 8.1 in 0.25% Tween 80 Curcurmin (DMSO), 1000 0.7 94.8 0.25% T80, 100 uM Ca- EDTA (RD174) Curcurmin (DMSO), 500 1.9 85.9 0.25% T80, 100 uM Ca- EDTA (RD174) Tween 80, 0.25% 0 15.1 11.9 Ca-EDTA (RD174), 100 0 3.2 76.5 m Tween 80 0.25% + Ca- 0 6.3 53.5 EDTA (RD174) 100 m DMSO (in de-ionized 0 12.8 4.9 water) 0.0736% Notes on above tables: .sup.1Treatments were amended into Potato Dextrose Agar (PDA) .sup.2Means were calculated based on 3 fungal isolates replicated 3 times, with 2 measurements per replicate (18 total measurements) .sup.3Means represent growth that occurred between 24 and 48 hours of incubation at 21 C. .sup.4% Inhibition calculated relative to non-amended control Mw of Baypure DS listed as 2,000-3,000, so based calculations on 2,500 g/mol.

Example 10

[0113] Experiments have indicated that the combination of EDDS trisodium salt and Mg-Chlorophyllin provides enhanced photodynamic suppression of bacterial growth (E. Coli) compared to each component individually.

TABLE-US-00012 TABLE 10 Results on inhibition in light (EDDS and MgChln) Treatment CFU/mL LogCFU/mL % Inactivation 50 mM EDDS 6.50E+05 5.81 88.1 10 M MgChln 1.30E+05 5.11 97.6 1 M MgChln 2.73E+05 5.44 95.0 10 M MgChln + <10000 <4 >99.8 50 mM EDDS 1 M MgChln + <10000 <4 >99.8 50 mM EDDS untreated control 5.47E+06 6.74

Example 11

[0114] Experiments have indicated that the combination of IDS (Baypure CX) and Mg-Chlorophyllin provides enhanced photodynamic suppression of bacterial growth (E. Coli) compared to each component individually.

TABLE-US-00013 TABLE 11 Results on inhibition in light (IDS and MgChln) Treatment CFU/mL LogCFU/mL % Inactivation 5 mM Baypure CX 2.09E+06 6.32 67.8 1 M MgChln 9.07E+05 5.96 86.1 1 M MgChln + 3.37E+05 5.53 94.8 5 mM Baypure CX Culture Control 6.50E+06 6.81

Example 12

[0115] Experiments have indicated that the combination of polyaspertate (Baypure DS) and Mg-Chlorophyllin provides enhanced photodynamic suppression of bacterial growth (E. Coli) compared to each component individually.

TABLE-US-00014 TABLE 12 Results on inhibition in light (polyaspertate and MgChln) Treatment CFU/mL LogCFU/mL % Inactivation Baypure DS (5%) 4.20E+06 6.62 40.3 1 M MgChln 6.13E+05 5.79 91.3 1 M MgChln + 6.67E+03 3.82 99.9 5% Baypure DS untreated control 7.03E+06 6.85

Example 13 (Curcuminnot a Nitrogen-Bearing Macrocyclic Compound)

[0116] Experiments have indicated that the combination of EDDS trisodium salt and Curcumin provides enhanced photodynamic suppression of bacteria (E. Coli) growth compared to each component individually. Curcumin solution was prepared by making fresh 100 mM stock in Arlasolve solvent and diluting in 0.25% Tween solution immediately before use.

TABLE-US-00015 TABLE 13 Results on inhibition in light (EDDS and Curcumin) Treatment CFU/mL LogCFU/mL % Inactivation 1 mM Curcumin in 0.25% 1.24E+06 6.09 86.0 Tween 80 solution 5.8% EDDS (50 mM) 1.05E+06 6.02 88.2 1 mM Curcumin in 0.25% <10000 <4 >99.8 Tween 80 solution + 50 mM EDDS untreated control 8.87E+06 6.95

Example 14

[0117] Control of dollar spot fungus (Sclerotinia homoeocarpa) was assessed for Mg Chlorophyllin, IDS (Baypure CX), and the combination thereof. Treatments were amended into Potato Dextrose Agar (PDA) at desired concentrations. Then, a 5 mm diameter plug of a Sclerotinia homoeocarpa isolate (3 isolates total tested) was inoculated into the center of the amended Petri-dish and incubated at 21 C. in the dark for 24 hours. After 24 hours, one set of Petri-dishes (in triplicate) was left in the dark and one set was placed under illumination for the remainder of the experiment (all at 21 C.). Radial growth of the fungus was monitored daily until the growth of S. homoeocarpa on non-amended PDA reached the edge of the Petri-dish. Illumination was provided by fluorescent lights emitting about 180 mol/m.sup.2/s photosynthetically active radiation (PAR).

TABLE-US-00016 TABLE 14A Results on inhibition in dark Mean Radial Rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.4 Non-amended 0 13.5 (control) Mg Chlorophyllin (Mg 31 9.1 32.5 Chl) Baypure CX (IDS) 500 12.7 6.2 Baypure CX (IDS) 1000 11.4 15.2 Baypure CX (IDS) 2000 9.3 31.3 Mg Chl + Baypure 31 + 500 9.2 32.1 CX Mg Chl + Baypure 31 + 1000 9.2 31.7 CX Mg Chl + Baypure 31 + 2000 7.9 41.2 CX

TABLE-US-00017 TABLE 14B Results on inhibition in light Mean Radial Rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.4 Non-amended 0 12.9 (control) Mg Chlorophyllin (Mg 31 5.3 59.2 Chl) Baypure CX (IDS) 500 11.8 8.6 Baypure CX (IDS) 1000 10.6 18.0 Baypure CX (IDS) 2000 8.9 30.9 Mg Chl + Baypure 31 + 500 4.5 65.2 CX Mg Chl + Baypure 31 + 1000 3.7 71.2 CX Mg Chl + Baypure 31 + 2000 3.1 76.0 CX Notes on above tables: .sup.1Treatments were amended into Potato Dextrose Agar (PDA); .sup.2Means were calculated based on 3 fungal isolates replicated 3 times, with 2 measurements per replicate (18 total measurements); .sup.3Means represent growth that occurred between 24 and 48 hours of incubation at 21 C.; .sup.4% Inhibition calculated relative to non-amended control.

Example 15

[0118] Control of dollar spot fungus (Sclerotinia homoeocarpa) was assessed for Mg Chlorophyllin, L-glutamic acid N,N-diacetic acid, tetrasodium salt (GLDA-NA.sub.4, Dissolvine GL-47-S), and the combination thereof. Treatments were amended into Potato Dextrose Agar (PDA) at desired concentrations. Then, a 5 mm diameter plug of a Sclerotinia homoeocarpa isolate (3 isolates total tested) was inoculated into the center of the amended Petri-dish and incubated at 21 C. in the dark for 24 hours. After 24 hours, one set of Petri-dishes (in triplicate) was left in the dark and one set was placed under illumination for the remainder of the experiment (all at 21 C.). Radial growth of the fungus was monitored daily until the growth of S. homoeocarpa on non-amended PDA reached the edge of the Petri-dish. Illumination was provided by fluorescent lights emitting about 180 mol/m.sup.2/s photosynthetically active radiation (PAR).

TABLE-US-00018 TABLE 15A Results on inhibition in dark Mean Radial Rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.4 Non-amended 0 13.5 (control) Mg Chlorophyllin (Mg 31 9.1 32.5 Chl) Dissolvine GL-47-S 500 13.2 2.1 (GLDA-Na4) Dissolvine GL-47-S 2500 4.6 66.3 (GLDA-Na4) Mg Chl + Dissolvine 31 + 500 10.1 25.5 GL-47-S (GLDA- Na4) Mg Chl + Dissolvine 31 + 2500 3.4 74.5 GL-47-S (GLDA- Na4)

TABLE-US-00019 TABLE 15B Results on inhibition in light Mean Radial Rate Growth % Treatment.sup.1 (M) (mm).sup.2,3 Inhibition.sup.4 Non-amended 0 12.9 (control) Mg Chlorophyllin (Mg 31 5.3 59.2 Chl) Dissolvine GL-47-S 500 12.1 6.4 (GLDA-Na4) Dissolvine GL-47-S 2500 3.4 73.8 (GLDA-Na4) Mg Chl + Dissolvine 31 + 500 4.4 66.1 GL-47-S (GLDA- Na4) Mg Chl + Dissolvine 31 + 2500 0.2 98.3 GL-47-S (GLDA- Na4) Notes on above tables: .sup.1Treatments were amended into Potato Dextrose Agar (PDA); .sup.2Means were calculated based on 3 fungal isolates replicated 3 times, with 2 measurements per replicate (18 total measurements); .sup.3Means represent growth that occurred between 24 and 48 hours of incubation at 21 C.; .sup.4% Inhibition calculated relative to non-amended control.

Example 16

[0119] In this example, control of dollar spot fungus (Sclerotinia homoeocarpa) with metallated chlorins of Type II was assessed. Treatments were amended into Potato Dextrose Agar (PDA) at desired concentrations. Then, a 5 mm diameter plug of a Sclerotinia homoeocarpa isolate (3 isolates total tested) was inoculated into the center of the amended Petri-dish and incubated at 21 C. in the dark for 24 hours. After 24 hours, one set of Petri-dishes (in triplicate) is left in the dark and one set is placed under illumination for the remainder of the experiment (all at 21 C.). Radial growth of the fungus is monitored daily until the growth of S. homoeocarpa on non-amended PDA reaches the edge of the Petri-dish. Illumination is provided by fluorescent lights emitting about 180 mol/m.sup.2/s photosynthetically active radiation (PAR).

TABLE-US-00020 TABLE 16A Results in dark Mean Radial Rate Growth Treatment.sup.1 (M) (mm).sup.23 % Inhibition.sup.5 Non-amended 0 13.5 (control) ZnCe6 5 13.2 2.47 ZnCe6 10 12.5 7.41 ZnCe6 31 10.3 23.46 AlCe6 5 13.8 1.85 AlCe6 10 13.0 3.70 AlCe6 31 13.1 3.09 SnCe6 5 13.2 2.47 SnCe6 10 13.0 3.70 SnCe6 31 10.8 19.75 PdCe6 5 11.5 18.82 PdCe6 10 9.0 36.47 PdCe6 31 8.1 42.75 Notes on above table: .sup.1Treatments were amended into Potato Dextrose Agar (PDA) .sup.2Means were calculated based on 3 fungal isolates replicated 3 times, with 2 measurements per replicate (18 total measurements) .sup.3Means represent growth that occurred between 24 and 48 hours of incubation at 21 C. .sup.5% Inhibition calculated relative to non-amended control

TABLE-US-00021 TABLE 16B Results on inhibition in light Mean Radial Chlorin Rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.5 Non-amended 0 12.8 (control) ZnCe6 5 11.5 10.4 ZnCe6 10 10.1 21.4 ZnCe6 31 7.5 41.6 AICe6 5 11.4 11.0 AICe6 10 7.5 41.6 AICe6 31 0.0 100.0 SnCe6 5 8.6 33.1 SnCe6 10 7.1 44.8 SnCe6 31 0.5 96.1 PdCe6 5 6.2 49.3 PdCe6 10 2.8 76.9 PdCe6 31 0 100.0 Notes on above table: .sup.1Treatments were amended into Potato Dextrose Agar (PDA) .sup.2Means were calculated based on 3 fungal isolates replicated 3 times, with 2 measurements per replicate (18 total measurements) .sup.3Means represent growth that occurred between 24 and 48 hours of incubation at 21 C. .sup.5% Inhibition calculated relative to non-amended control
Tables 16A and 16B show that various metallated chlorophyllins of type II inhibited dollar spot fungus in the presence of light.

Example 17Comparative Example

[0120] In this example, control of dollar spot fungus (Sclerotinia homoeocarpa) with a cobalt chlorins (which is not a Type II photosensitizer) was assessed. Treatments were amended into Potato Dextrose Agar (PDA) at desired concentrations. Then, a 5 mm diameter plug of a Sclerotinia homoeocarpa isolate (3 isolates total tested) was inoculated into the center of the amended Petri-dish and incubated at 21 C. in the dark for 24 hours. After 24 hours, one set of Petri-dishes (in triplicate) is left in the dark and one set is placed under illumination for the remainder of the experiment (all at 21 C.). Radial growth of the fungus is monitored daily until the growth of S. homoeocarpa on non-amended PDA reaches the edge of the Petri-dish. Illumination is provided by fluorescent lights emitting about 180 mol/m.sup.2/s photosynthetically active radiation (PAR).

TABLE-US-00022 TABLE 17A Results in dark Mean Radial Rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.5 Non-amended 0 13.6 (control) CoCe6 10 13.3 2.05 CoCe6 31 12.9 4.92

TABLE-US-00023 TABLE 17B Results on inhibition in light Mean Radial Rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.5 Non-amended 0 12.8 (control) CoCe6 10 12.4 3.0 CoCe6 31 12.3 3.9 Notes on above tables: .sup.1Treatments were amended into Potato Dextrose Agar (PDA) .sup.2Means were calculated based on 3 fungal isolates replicated 3 times, with 2 measurements per replicate (18 total measurements) .sup.3Means represent growth that occurred between 24 and 48 hours of incubation at 21 C. .sup.5% Inhibition calculated relative to non-amended control

[0121] In this example, control of dollar spot fungus (Sclerotinia homoeocarpa) with Cu chlorophyllin was assessed. Treatments were amended into Potato Dextrose Agar (PDA) at desired concentrations. Then, a 5 mm diameter plug of a Sclerotinia homoeocarpa isolate (5 isolates total tested) was inoculated into the center of the amended Petri-dish and incubated at 21 C. in the dark for 24 hours. After 24 hours, one set of Petri-dishes (in triplicate) is left in the dark and one set is placed under illumination for the remainder of the experiment (all at 21 C.). Radial growth of the fungus is monitored daily until the growth of S. homoeocarpa on non-amended PDA reaches the edge of the Petri-dish. Illumination is provided by fluorescent lights emitting about 180 mol/m.sup.2/s photosynthetically active radiation (PAR).

TABLE-US-00024 TABLE 17C Results in dark Mean Radial Rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.5 Non-amended 0 13.87 (control) Cu Chlorophyllin 31.62 14.30 3.13 (CuChln) CuChln 100 14.33 3.37 CuChln 316.2 14.60 5.29 CuChln 1000 14.40 3.85

TABLE-US-00025 TABLE 17D Results on inhibition in light Mean Radial Rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.5 Non-amended 0 11.80 (control) Cu Chlorophyllin 31.62 14.03 18.93 (CuChln) CuChln 100 14.20 20.34 CuChln 316.2 14.93 26.55 CuChln 1000 15.20 28.81 Notes on above table: .sup.1Treatments were amended into Potato Dextrose Agar (PDA) .sup.2Means were calculated based on 3 fungal isolates replicated 3 times, with 2 measurements per replicate (18 total measurements) .sup.3Means represent growth that occurred between 24 and 48 hours of incubation at 21 C. .sup.5% Inhibition calculated relative to non-amended control
Tables 17A-17D show that metallated chlorophyllins that are not type II photosensitizersor in other words, metallated chlorophyllins that do not generate singlet oxygens in the presence of lightdo not inhibit dollar spot fungus whether in the presence or absence of light.

Example 18

[0122] In this example, control of dollar spot fungus (Sclerotinia homoeocarpa) with chlorins combined with a chelating agent was assessed. Treatments were prepared in Phosphate Buffered Saline (PBS) in 24 well plates (in duplicates for light vs. dark incubation) at desired concentrations. Then, a 5 mm diameter plug of a Sclerotinia homoeocarpa isolate (3 isolates total tested) was inoculated into the PBS and incubated at 21 C. in the dark for 2 hours. After 2 hours, one of the 24 well plates (with isolates in triplicate) is left in the dark and one 24 well plate is placed under illumination for 1 hour (all at 21 C.). Following illumination, fungal plugs are removed from PBS, blotted dry on sterile filter paper and transferred to non-amended Potato Dextrose Agar (PDA). Radial growth of the fungus is monitored daily until the growth of S. homoeocarpa reaches the edge of the Petri-dish. Illumination is provided by LED lights emitting about 1000 mol/m.sup.2/s photosynthetically active radiation (PAR).

TABLE-US-00026 TABLE 18A Results in dark Mean Radial Rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.5 PBS (control) 0 11.17 EDTA, disodium, 500 6.72 39.80 dihydrate (NaEDTA) Mg chlorophyllin 100 12.50 11.94 (Mg Chln) Mg Chln 10 11.61 3.98 Ce6 100 10.78 3.48 Ce6 10 11.33 1.49 NaEDTA + MgChln 500 + 100 8.06 27.86 NaEDTA + MgChln 500 + 10 8.17 26.87 NaEDTA + Ce6 500 + 100 7.17 35.82 NaEDTA + Ce6 500 + 10 5.44 51.24 Notes on above table: .sup.1Treatments were prepared in Phosphate Buffered Saline (PBS), incubated on shaker (200 rpm) for 2 hours in the dark, then exposed to light (Helios, 1000 PAR) or dark for 1 hour. .sup.2Means were calculated based on 3 fungal isolates replicated 3 times, with 2 measurements per replicate (18 total measurements) .sup.3Means represent growth that occurred between 24 and 48 hours of incubation at 21 C. .sup.5% Inhibition calculated relative to non-amended control

TABLE-US-00027 TABLE 18B Results on inhibition in light Mean Radial Rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.5 PBS (control) 0 11.06 EDTA, disodium, 500 9.22 16.58 dihydrate (NaEDTA) Mg chlorophyllin (Mg 100 8.56 22.61 Chln) Mg Chln 10 7.56 31.66 Ce6 100 9.83 11.06 Ce6 10 8.39 24.12 NaEDTA + MgChln 500 + 100 2.50 77.39 NaEDTA + MgChln 500 + 10 1.06 90.45 NaEDTA + Ce6 500 + 100 2.61 76.38 NaEDTA + Ce6 500 + 10 0.94 91.46 Notes on above table: .sup.1Treatments were prepared in Phosphate Buffered Saline (PBS), incubated on shaker (200 rpm) for 2 hours in the dark, then exposed to light (Helios, 1000 PAR) or dark for 1 hour. .sup.2Means were calculated based on 3 fungal isolates replicated 3 times, with 2 measurements per replicate (18 total measurements) .sup.3Means represent growth that occurred between 24 and 48 hours of incubation at 21 C. .sup.5% Inhibition calculated relative to non-amended control

Example 19

[0123] In this example, control of dollar spot fungus (Sclerotinia homoeocarpa) with Zinc Protoporphyrin IX (Zn-PPIX) combined with EDTA was assessed. Treatments were prepared in Phosphate Buffered Saline (PBS) in 24 well plates (in duplicates for light vs. dark incubation) at desired concentrations. Then, a 5 mm diameter plug of a Sclerotinia homoeocarpa isolate (3 isolates total tested) was inoculated into the PBS and incubated at 21 C. in the dark for 2 hours. After 2 hours, one of the 24 well plates (with isolates in triplicate) is left in the dark and one 24 well plate is placed under illumination for 1 hour (all at 21 C.). Following illumination, fungal plugs are removed from PBS, blotted dry on sterile filter paper and transferred to non-amended Potato Dextrose Agar (PDA). Radial growth of the fungus is monitored daily until the growth of S. homoeocarpa reaches the edge of the Petri-dish. Illumination is provided by LED lights emitting about 1000 mol/m.sup.2/s photosynthetically active radiation (PAR).

TABLE-US-00028 TABLE 19A Results on inhibition in light Mean Radial Rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.5 PBS (control) 0 11.11 EDTA, disodium, 500 7.11 36.00 dihydrate (NaEDTA) Zn Protoporphyrin IX 10 10.28 7.50 (PPIX) Notes on above table: .sup.1Treatments were prepared in Phosphate Buffered Saline (PBS), incubated on shaker (200 rpm) for 2 hours in the dark, then exposed to light (Helios, 1000 PAR) or dark for 1 hour. .sup.2Means were calculated based on 3 fungal isolates replicated 3 times, with 2 measurements per replicate (18 total measurements) .sup.3Means represent growth that occurred between 24 and 48 hours of incubation at 21 C. .sup.5% Inhibition calculated relative to non-amended control

Example 20

[0124] In this example, control of dollar spot fungus (Sclerotinia homoeocarpa) with bacteriochlorin combined with EDTA was assessed. Treatments were prepared in Phosphate Buffered Saline (PBS) in 24 well plates (in duplicates for light vs. dark incubation) at desired concentrations. Then, a 5 mm diameter plug of a Sclerotinia homoeocarpa isolate (3 isolates total tested) was inoculated into the PBS and incubated at 21 C. in the dark for 2 hours. After 2 hours, one of the 24 well plates (with isolates in triplicate) is left in the dark and one 24 well plate is placed under illumination for 1 hour (all at 21 C.). Following illumination, fungal plugs are removed from PBS, blotted dry on sterile filter paper and transferred to non-amended Potato Dextrose Agar (PDA). Radial growth of the fungus is monitored daily until the growth of S. homoeocarpa reaches the edge of the Petri-dish. Illumination is provided by LED lights emitting about 1000 mol/m.sup.2/s photosynthetically active radiation (PAR).

TABLE-US-00029 TABLE 20A Results in dark Mean Radial Rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.5 PBS (control) 0 10.67 Bacteriochlorin 1 10.94 2.60 (BchlNA) (in Pluronics + Arlasolve) BchlNa (in Pluronics + 10 11.39 6.77 Arlasolve) NaEDTA + BchlNa 500 + 1 6.78 36.46 NaEDTA + BchlNa 500 + 10 5.06 52.60 NaEDTA 500 7.17 32.81 Notes on above table: .sup.1Treatments were prepared in Phosphate Buffered Saline (PBS), incubated on shaker (200 rpm) for 2 hours in the dark, then exposed to light (Helios, 1000 PAR) or dark for 1 hour. .sup.2Means were calculated based on 3 fungal isolates replicated 3 times, with 2 measurements per replicate (18 total measurements) .sup.3Means represent growth that occurred between 24 and 48 hours of incubation at 21 C. .sup.5% Inhibition calculated relative to non-amended control

TABLE-US-00030 TABLE 20B Results on inhibition in light Mean Radial Rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.5 PBS (control) 0 10.78 Bacteriochlorin 1 10.44 3.09 (BchlNA) (in Pluronics + Arlasolve) BchlNa (in Pluronics + 10 8.33 22.68 Arlasolve) NaEDTA + BchlNa 500 + 1 4.72 56.19 NaEDTA + BchlNa 500 + 10 0.67 93.81 NaEDTA 500 7.17 33.51 Notes on above table: .sup.1Treatments were prepared in Phosphate Buffered Saline (PBS), incubated on shaker (200 rpm) for 2 hours in the dark, then exposed to light (Helios, 1000 PAR) or dark for 1 hour. .sup.2Means were calculated based on 3 fungal isolates replicated 3 times, with 2 measurements per replicate (18 total measurements) .sup.3Means represent growth that occurred between 24 and 48 hours of incubation at 21 C. .sup.5% Inhibition calculated relative to non-amended control

Example 21

[0125] In this example, control of dollar spot fungus (Sclerotinia homoeocarpa) with Aluminum chlorin e6 combined with EDTA was assessed. Treatments were prepared in Phosphate Buffered Saline (PBS) in 24 well plates (in duplicates for light vs. dark incubation) at desired concentrations. Then, a 5 mm diameter plug of a Sclerotinia homoeocarpa isolate (3 isolates total tested) was inoculated into the PBS and incubated at 21 C. in the dark for 2 hours. After 2 hours, one of the 24 well plates (with isolates in triplicate) is left in the dark and one 24 well plate is placed under illumination for 1 hour (all at 21 C.). Following illumination, fungal plugs are removed from PBS, blotted dry on sterile filter paper and transferred to non-amended Potato Dextrose Agar (PDA). Radial growth of the fungus is monitored daily until the growth of S. homoeocarpa reaches the edge of the Petri-dish. Illumination is provided by LED lights emitting about 1000 mol/m.sup.2/s photosynthetically active radiation (PAR).

TABLE-US-00031 TABLE 21A Results in dark Mean Radial Rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.5 PBS (control) 0 12.5 Al Ce6 1 13 4 Al Ce6 10 13.17 5.33 EDTA disodium, 500 11.06 11.56 dihydrate (NaEDTA) NaEDTA + Al Ce6 500 + 1 11.78 5.78 NaEDTA + Al Ce6 500 + 10 9.39 24.89 Notes on above table: .sup.1Treatments were prepared in Phosphate Buffered Saline (PBS), incubated on shaker (200 rpm) for 2 hours in the dark, then exposed to light (Helios, 1000 PAR) or dark for 1 hour. .sup.2Means were calculated based on 3 fungal isolates replicated 3 times, with 2 measurements per replicate (18 total measurements) .sup.3Means represent growth that occurred between 24 and 48 hours of incubation at 21 C. .sup.5% Inhibition calculated relative to non-amended control

TABLE-US-00032 TABLE 21B Results on inhibition in light Mean Radial Rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.5 PBS (control) 0 12.72 Al Ce6 1 13 2.18 Al Ce6 10 4.78 62.45 EDTA disodium, 500 10.78 15.28 dihydrate (NaEDTA) NaEDTA + Al Ce6 500 + 1 6.39 49.78 NaEDTA + Al Ce6 500 + 10 3 76.42 Notes on above table: .sup.1Treatments were prepared in Phosphate Buffered Saline (PBS), incubated on shaker (200 rpm) for 2 hours in the dark, then exposed to light (Helios, 1000 PAR) or dark for 1 hour. .sup.2Means were calculated based on 3 fungal isolates replicated 3 times, with 2 measurements per replicate (18 total measurements) .sup.3Means represent growth that occurred between 24 and 48 hours of incubation at 21 C. .sup.5% Inhibition calculated relative to non-amended control

Example 22

[0126] In this example, control of dollar spot fungus (Sclerotinia homoeocarpa) with hematoporphyrin IX dichloride combined with a chelating agent (EDTA disodium, dihydrate) was assessed. Treatments were prepared in Phosphate Buffered Saline (PBS) in 24 well plates (in duplicates for light vs. dark incubation) at desired concentrations. Then, a 5 mm diameter plug of a Sclerotinia homoeocarpa isolate (3 isolates total tested) was inoculated into the PBS and incubated at 21 C. in the dark for 2 hours. After 2 hours, one of the 24 well plates (with isolates in triplicate) is left in the dark and one 24 well plate is placed under illumination for 1 hour (all at 21 C.). Following illumination, fungal plugs are removed from PBS, blotted dry on sterile filter paper and transferred to non-amended Potato Dextrose Agar (PDA). Radial growth of the fungus is monitored daily until the growth of S. homoeocarpa reaches the edge of the Petri-dish. Illumination is provided by LED lights emitting about 1000 mol/m.sup.2/s photosynthetically active radiation (PAR).

TABLE-US-00033 TABLE 22A Results in dark Mean Radial Rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.5 PBS 0 14.28 Hematoporphyrin IX 10 15.06 5.45 dichloride (HemIX) EDTA disodium, 500 14.83 3.89 dihydrate (NaEDTA) HemIX + NaEDTA 10 + 500 14.78 3.50 Notes on above table: .sup.1Treatments were prepared in Phosphate Buffered Saline (PBS), incubated on shaker (200 rpm) for 2 hours in the dark, then exposed to light (Helios, 1000 PAR) or dark for 1 hour. .sup.2Means were calculated based on 3 fungal isolates replicated 3 times, with 2 measurements per replicate (18 total measurements) .sup.3Means represent growth that occurred between 48 and 72 hours of incubation at 21 C. .sup.5% Inhibition calculated relative to non-amended control

TABLE-US-00034 TABLE 22B Results on inhibition in light Mean Radial Rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.5 PBS 0 13.72 Hematoporphyrin IX 10 8.39 38.87 dichloride (HemIX) EDTA disodium, 500 8.89 35.22 dihydrate (NaEDTA) HemIX + NaEDTA 10 + 500 0.56 95.95 Notes on above table: .sup.1Treatments were prepared in Phosphate Buffered Saline (PBS), incubated on shaker (200 rpm) for 2 hours in the dark, then exposed to light (Helios, 1000 PAR) or dark for 1 hour. .sup.2Means were calculated based on 3 fungal isolates replicated 3 times, with 2 measurements per replicate (18 total measurements) .sup.3Means represent growth that occurred between 48 and 72 hours of incubation at 21 C. .sup.5% Inhibition calculated relative to non-amended control

Example 23Test on Host Plant Nicotiana benthamiana

[0127] In this example, control of the fungal plant pathogen Colletotrichum orbiculare (Cgm) on the host plant Nicotiana benthamiana following treatment with Mg Chlorophyllin combined with a chelating agent and an emulsifier was assessed. Treatments were applied to N. benthamiana plants approximately 2 hours prior to inoculation with a spore suspension of Cgm. Plants were then exposed to light for a 24 hour period followed by dark incubation until disease symptoms were evident on the water treated control plants. Once disease symptoms were evident, lesions were counted and leaf area measured in order to determine the number of lesions/cm.sup.2 leaf area. Four replicate plants were used per treatment and plants were randomized under the light source. Illumination is provided by LED lights emitting about 180 mol/m.sup.2/s photosynthetically active radiation (PAR).

TABLE-US-00035 TABLE 23 Results on inhibition in light Lesions/cm.sup.2 % Treatment.sup.1 Leaf Area.sup.2 Inhibition.sup.3 Phytotoxicity.sup.4 0.1% Mg Chlorophyllin + 0.146 97.21 0 0.025% Ca EDTA, disodium + 0.0175% Atlox AL-3273 0.025% Ca EDTA, 1.857 64.35 0 disodium + 0.0175% Atlox AL-3273 water control 5.210 0 .sup.1Treatments were applied ~2 hours prior to inoculation with Cgm. .sup.2Numbers are means of 4 replications. .sup.3% Inhibition calculated relative to non-amended control. .sup.4Phytotoxicity ratings: 0 = healthy plant, no damage; 3 = slight damage; 7 = severe damage; 10 = dead plant. Less than 3 is considered acceptable

Example 24

[0128] In this example, control of the fungal plant pathogen Colletotrichum orbiculare (Cgm) on the host plant Nicotiana benthamiana following treatment with Ce6 trisodium salt (Ce6Na3) combined with a polyalphaolefin (PAO) oil was assessed. Treatments were applied to N. benthamiana plants approximately 2 hours prior to inoculation with a spore suspension of Cgm. Plants were then exposed to light for a 24 hour period followed by dark incubation until disease symptoms were evident on the water treated control plants. Once disease symptoms were evident, lesions were counted and leaf area measured in order to determine the number of lesions/cm.sup.2 leaf area. Four replicate plants were used per treatment and plants were randomized under the light source. Illumination is provided by LED lights emitting about 180 mol/m.sup.2/s photosynthetically active radiation (PAR).

TABLE-US-00036 TABLE 24 Results on inhibition in light Lesions/cm.sup.2 % Treatment.sup.1 Leaf Area.sup.2 Inhibition.sup.4 Phytotoxicity.sup.5 0.1% Ce6Na3 + 0.25% 0.64 97.21 2 PAO 4 Cst (contains 7% Atlox AL-3273) 0.25% PAO 4 Cst (contains 2.06 64.35 0 7% Atlox AL-3273) water control 3.59 0 1Treatments were applied ~2 hours prior to inoculation with Cgm. .sup.2Numbers are means of 4 replications. .sup.3Lesions/cm.sup.2 Leaf Area followed by a letter in common are not statistically different (p = 0.05) .sup.4% Inhibition calculated relative to non-amended control. .sup.5Phytotoxicity ratings: 0 = healthy plant, no damage; 3 = slight damage; 7 = severe damage; 10 = dead plant. Less than 3 is considered acceptable

Example 25

[0129] In this example, control of dollar spot fungus (Sclerotinia homoeocarpa) with Mg Chlorophyllins combined with a chelating agent (Tannic acid) was assessed. Treatments were prepared in Phosphate Buffered Saline (PBS) in 24 well plates (in duplicates for light vs. dark incubation) at desired concentrations. Then, a 5 mm diameter plug of a Sclerotinia homoeocarpa isolate (3 isolates total tested) was inoculated into the PBS and incubated at 21 C. in the dark for 2 hours. After 2 hours, one of the 24 well plates (with isolates in triplicate) is left in the dark and one 24 well plate is placed under illumination for 1 hour (all at 21 C.). Following illumination, fungal plugs are removed from PBS, blotted dry on sterile filter paper and transferred to non-amended Potato Dextrose Agar (PDA). Radial growth of the fungus is monitored daily until the growth of S. homoeocarpa reaches the edge of the Petri-dish. Illumination is provided by LED lights emitting about 1000 mol/m.sup.2/s photosynthetically active radiation (PAR).

TABLE-US-00037 TABLE 25A Results in dark Mean Radial Rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.5 PBS 0 10.67 Tannic acid-500 500 11.33 6.25 Tannic acid-1500 1500 9.89 7.29 Mg Chl-5 5 10.56 1.04 MgChl + Tannic 5 + 500 11.50 7.81 Acid-5 + 500 MgChl + Tannic 5 + 1500 9.50 10.94 Acid-5 + 1500 Notes on above table: .sup.1Treatments were prepared in Phosphate Buffered Saline (PBS), incubated on shaker (200 rpm) for 2 hours in the dark, then exposed to light (Helios, 1000 PAR) or dark for 1 hour. .sup.2Means were calculated based on 3 fungal isolates replicated 3 times, with 2 measurements per replicate (18 total measurements) .sup.3Means represent growth that occurred between 24 and 48 hours of incubation at 21 C. .sup.5% Inhibition calculated relative to non-amended control

TABLE-US-00038 TABLE 25B Results on inhibition in light Mean Radial Rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.5 PBS 0 10.89 Tannic acid-500 500 11.94 9.69 Tannic acid-1500 1500 9.11 16.33 Mg Chl-5 5 6.78 37.76 MgChl + Tannic 5 + 500 6.39 41.33 Acid-5 + 500 MgChl + Tannic 5 + 1500 2.50 77.04 Acid-5 + 1500 Notes on above table: .sup.1Treatments were prepared in Phosphate Buffered Saline (PBS), incubated on shaker (200 rpm) for 2 hours in the dark, then exposed to light (Helios, 1000 PAR) or dark for 1 hour. .sup.2Means were calculated based on 3 fungal isolates replicated 3 times, with 2 measurements per replicate (18 total measurements) .sup.3Means represent growth that occurred between 24 and 48 hours of incubation at 21 C. .sup.5% Inhibition calculated relative to non-amended control
Combining MgChln with Tannic acid provided greater inhibition on fungal growth than using MgChln or Tannic acid alone.

Example 26

[0130] In this example, application of a porphyrin photosensitizer compound and chelating agents suppressed growth of Bacteria (Erwinia amylovora) that causes Fire Blight disease using photodynamic inhibition. In particular, Mg-chlorophyllin and disodium EDTA and Baypre DS100 were used and their combination was shown to inactivate E. amylovora growth. In the experiment, E. amylovora were grown in liquid medium. Samples were incubated for 30 min with 100 M of Mg-Chlorophyllin with or without Chelators, and then illuminated with 395 nm (fluence 26.6 J cm.sup.2) for 30 mins. CFU of the bacteria was counted on the plates. The following table summarizes the results:

TABLE-US-00039 TABLE 26 Results on relative inactivation of bacteria after PDI treatment Relative Log Relative inactivation inactivation Treatment [CFU.sub.Co//CFU.sub.sample] [CFU.sub.Co//CFU.sub.sample] Untreated control, 1 0 without light no light, 100 M 8.30E+00 0.919078 Chlorophyllin + 1.2% BaypureDS100 With light, 100 M 1.40E+07 7.146128 Chlorophyllin + 1.2% BaypureDS100 no light, 100 M 5.30E+04 4.724276 Chlorophyllin + 1.2% BaypureDS100, 1 mM Na2EDTA With light, 100 M >1.6E+08 >8 Chlorophyllin + 1.2% BaypureDS100, 1 mM Na2EDTA
Combining EDTA and Baypure DS100 with chlorophyllin increased the antibacterial effect significantly. E. amylovora was almost completely inactivated with the combination of the three compounds under the test conditions.

Example 27

[0131] In this example, application of a porphyrin photosensitizer compound and a chelating agent suppressed growth of Botrytis cinerea fungi that causes Gray Mold disease using photodynamic inhibition. In particular, Mg-chlorophyllin and sodium polyaspartate (Baypure DS 100) were used, and the combination of the porphyrin photosensitizer compound and each chelating agent was shown to inactivate Botrytis cinerea growth.

[0132] In the experiments, Botrytis cinerea mycelia were grown in liquid medium for 48 hours. Small spheres of the mycelia (average diameter 2 mm) were incubated for 100 minutes with Mg-Chlorophyllin with or without Baypure DS100 Samples were illuminated with 395 nm (fluence 106.6 J cm.sup.2) for 120 min and the radial growth of mycelial patches after 7 days on agar medium was measured. A sample was considered dead, if there was no growth observable after 7 days on agar plates. The following table summarizes the results:

TABLE-US-00040 TABLE 27 Results on percentage of dead B. cinereal mycelia Treatment Percentage of dead samples [%] Untreated control in dark 0 BaypureDS100 1.2%, without light 0 BaypureDS100 1.2%, with light 0 MgChln 100 M, with light 0 MgChln 100 pM, and 1.2% 44.4 BaypureDS100, with light

[0133] The phototreatment of chlorophyllin or Baypure alone had no effect on fungal mycelia. Adding Baypure DS100 into Chlorophyllin increased the suppression on the growth of fungal mycelia, notably at Chlorophyllin concentrations of 100 M at the test conditions. Botrytis cinerea was greatly inactivated when treated by combination of 100 M Chlorophyllin and 1.2% Baypure DS100.

Example 28Test on Apple Trees

[0134] In this example, a porphyrin photosensitizer compound and a chelating agent were applied in the field to suppress growth of Bacteria (Erwinia amylovora) that causes Fire Blight disease on apple trees (Gala). In particular, Mg-chlorophyllin and disodium EDTA with and without Baypure100DS were used and their combination was shown to suppress the apple fire blight in the field condition. No phytotoxicity from the treatments were observed on the apple trees.

[0135] Erwinia amylovora was inoculated at the 80% of bloom of the apple trees. Mg-chlorophyllin and the chelator combinations were applied three times (2 hrs before inoculation, and 24 hrs, 72 hrs after inoculation) at the spray volume of 300 gal per acre. Fire Blight (Blossom blight) disease infections were rated several weeks after inoculation. The average disease infection (%) is shown in table below.

TABLE-US-00041 TABLE 28 Disease Percent of Treatments infection, % control, % Phytotoxicity .sup.1 Untreated control 37.1 0% 0 0.2% MgChln + 11.4 69% 0 0.1% NaEDTA + 0.05% Atlox AL- 3273 + 0.05% Saponin 0.2% MgChln + 15.3 59% 0 1.2% Baypure + 0.05% NaEDTA + 0.05% Atlox AL- 3273 + 0.05% Saponin .sup.1 Phytotoxicity ratings: 0 = healthy plant, no damage; 3 = slight damage; 7 = severe damage; 10 = dead plant. Less than 3 is considered acceptable

Example 29Test on Tomato Plants

[0136] In this example, application of a porphyrin photosensitizer compound and a chelating agent suppressed growth of fungal pathogen Alternaria solani that cause Early blight on Tomato plants. In particular, Mg-chlorophyllin and disodium EDTA with and without Baypure100DS were used and their combination was shown to suppress Early blight on tomato in the field condition. No phytotoxicity from the treatments were observed on the tomato plants.

[0137] Alternaria solani was inoculated twice on the tomato plants at 35 days and 42 days after transplanting. Mg-chlorophyllin and the chelator combinations were applied total 6 times (2 hrs before 1.sup.st inoculation, 24 hrs after 1.sup.st inoculation, followed by 3 more application at 7 days intervals) at the spray volume of 50 gal per acre. Early blight disease infections were rated 11 days after 2.sup.nd inoculation. The average disease infection (%) and the standard area under the disease progress curve (SAUDPC) are shown in the table below.

TABLE-US-00042 TABLE 29 Disease Percent of Phyto- Treatments severity, % SAUDPC control, % toxicity .sup.1 Untreated control 0.93 0.350 0 0 0.2% MgChln + 0.84 0.219 39.6% 0 0.1% NaEDTA + 0.05% Atlox AL- 3273 + 0.05% Saponin 0.2% MgChln + 0.59 0.128 63.4% 0 1.2% Baypure + 0.05% NaEDTA + 0.05% Atlox AL- 3273 + 0.05% Saponin .sup.1 Phytotoxicity ratings: 0 = healthy plant, no damage; 3 = slight damage; 7 = severe damage; 10 = dead plant. Less than 3 is considered acceptable

Example 30Test on Strawberry Plants

[0138] To evaluate the phytotoxicity of photosensitizers with chelators on plants, MgChln and Na2EDTA combination was applied on strawberry plants (Fragaria vesa) to test whether the treatment cause leaf damage.

[0139] Treatments were sprayed onto seed strawberry plants derived from seeds and grown outside under a transparent rain cover. Total three applications were made in 7-day interval. Phytotoxiciy on the plants were assessed on day 0 and day 21. No leaf damage was observed.

TABLE-US-00043 TABLE 30 Phytotoxicity Phytotoxicity .sup.1 Treatments Day 0 Day 21 Untreated control 0 0 Water alone 0 0 100 M MgChln + 5 mM Na.sub.2EDTA 0 0 .sup.1 Phytotoxicity ratings: 0 = healthy plant, no damage; 3 = slight damage; 7 = severe damage; 10 = dead plant. Less than 3 is considered acceptable

Example 31

[0140] In this example, control of dollar spot fungus (Sclerotinia homoeocarpa) with meso-Tetra (N-methyl-4-pyridyl) porphine tetra tosylate (cationic porphyrin) combined with a chelating agent (EDTA, disodium, dihydrate) was assessed. Treatments were prepared in Phosphate Buffered Saline (PBS) in 24 well plates (in duplicates for light vs. dark incubation) at desired concentrations. Then, a 5 mm diameter plug of a Sclerotinia homoeocarpa isolate (3 isolates total tested) was inoculated into the PBS and incubated at 21 C. in the dark for 2 hours. After 2 hours, one of the 24 well plates (with isolates in triplicate) is left in the dark and one 24 well plate is placed under illumination for 1 hour (all at 21 C.). Following illumination, fungal plugs are removed from PBS, blotted dry on sterile filter paper and transferred to non-amended Potato Dextrose Agar (PDA). Radial growth of the fungus is monitored daily until the growth of S. homoeocarpa reaches the edge of the Petri-dish. Illumination is provided by LED lights emitting about 1000 mol/m.sup.2/s photosynthetically active radiation (PAR).

TABLE-US-00044 TABLE 31A Results in dark Mean Radial Rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.5 PBS 0 14.67 EDTA, disodium, 500 12.67 13.64 dihydrate (NaEDTA) meso-Tetra (N-methyl-4- 0.1 14.06 4.17 pyridyl) porphine tetra tosylate meso-Tetra (N-methyl-4- 0.1 + 500 13.61 7.20 pyridyl) porphine tetra tosylate + NaEDTA Notes on above table: .sup.1Treatments were prepared in Phosphate Buffered Saline (PBS), incubated on shaker (200 rpm) for 2 hours in the dark, then exposed to light (Helios, 1000 PAR) or dark for 1 hour. .sup.2Means were calculated based on 3 fungal isolates replicated 3 times, with 2 measurements per replicate (18 total measurements) .sup.3Means represent growth that occurred between 24 and 48 hours of incubation at 21 C. .sup.5% Inhibition calculated relative to non-amended control

TABLE-US-00045 TABLE 31B Results on inhibition in light Mean Radial Rate Growth Treatment.sup.1 (M) (mm).sup.2,3 % Inhibition.sup.5 PBS 0 12.83 EDTA, disodium, 500 8.17 36.36 dihydrate (NaEDTA) meso-Tetra (N-methyl-4- 0.1 8.39 34.63 pyridyl) porphine tetra tosylate meso-Tetra (N-methyl-4- 0.1 + 500 2.89 77.49 pyridyl) porphine tetra tosylate + NaEDTA Notes on above table: .sup.1Treatments were prepared in Phosphate Buffered Saline (PBS), incubated on shaker (200 rpm) for 2 hours in the dark, then exposed to light (Helios, 1000 PAR) or dark for 1 hour. .sup.2Means were calculated based on 3 fungal isolates replicated 3 times, with 2 measurements per replicate (18 total measurements) .sup.3Means represent growth that occurred between 24 and 48 hours of incubation at 21 C. .sup.5% Inhibition calculated relative to non-amended control

Example 32

[0141] In this example, control of the fungal plant pathogen Colletotrichum orbiculare (Cgm) on the host plant Nicotiana benthamiana following treatment with Mg Chlorophyllin combined with a chelating agent was assessed. Treatments were applied to N. benthamiana plants approximately 2 hours prior to inoculation with a spore suspension of Cgm. Plants were then exposed to light for a 12 hour period followed by dark incubation until disease symptoms were evident on the water treated control plants. Once disease symptoms were evident, lesions were counted and leaf area measured in order to determine the number of lesions/cm.sup.2 leaf area. Four replicate plants were used per treatment and plants were randomized under the light source. Illumination is provided by LED lights emitting about 180 mol/m.sup.2/s photosynthetically active radiation (PAR).

TABLE-US-00046 TABLE 32 Lesions/cm.sup.2 % Treatment.sup.1 Leaf Area.sup.2 Inhibition.sup.4 Phytotoxicity .sup.5 0.01% Mg Chlorophyllin + 4.60 52.3 0 0.0175% Atlox AL-3273 0.05% Na2EDTA + 0.0175% 6.90 28.5 0 Atlox AL-3273 0.01% Mg Chlorophyllin + 0.56 94.2 0 0.05% Na2EDTA, + 0.0175% Atlox AL-3273 water control 9.64 0 .sup.1Treatments were applied ~2 hours prior to inoculation with Cgm. .sup.2Numbers are means of 4 replications. .sup.4% Inhibition calculated relative to non-amended control. .sup.5 Phytotoxicity ratings: 0 = healthy plant, no damage; 3 = slight damage; 7 = severe damage; 10 = dead plant. Less than 3 is considered acceptable

Additional Information on Experimentation

[0142] The protocols for the photodynamic inhibition (PDI) of A. solani and B. cinerea differ in the way the spheres of mycelia were grown. The PDI treatment itself and evaluation are performed in the same manner.

[0143] The protocol for the PDI of A. solani was as follows (for Experiment 2 and 5): [0144] Grow A. solani on tomato-agar for some days. [0145] Transfer parts of the growing mycelium form the edge of the A. solani (no conidia) to liquid tomato medium. [0146] Incubate overnight at 26 C. under constant agitation. [0147] Transfer the grown mycelia (spheres of mycelia, approximately 2 mm in diameter) to 24 well plates. [0148] Add 500 microlitres of the PS to the well (PBS for the Co and light only controls). [0149] Incubate under agitation for 100 minutes. [0150] Illuminate under agitation for 120 minutes. [0151] Transfer the treated mycelia to tomato-agar plates and incubate for one week at room temperature. [0152] Evaluate by counting of dead samples.

[0153] The protocol for the PDI of B. cinerea was as follows (for experiments 3 and 4 described above): [0154] Grow B. cinerea on malt-peptone-agar for some days. [0155] Transfer spores of B. cinerea to liquid malt-peptone medium. [0156] Incubate for 2-3 days at 26 C. under constant agitation. [0157] Transfer the grown mycelia (spheres of mycelia, approximately 2 mm diameter) to 24 well plates. [0158] Add 500 l of the PS to the well. [0159] Incubate under agitation for 100 minutes in the dark. [0160] Illuminate under agitation for 120 minutes. [0161] Transfer the treated mycelia to malt-peptone-agar plates and incubate for one week at room temperature. [0162] Evaluate by counting of dead samples.

[0163] Regarding the E. Coli assay tests in the above examples, the following procedure was used: [0164] 1. E. Coli K-12 inoculated in 5 mL TSB and placed in a shaking incubator overnight. [0165] 2. Dilute culture in 500 mL PBS (108 CFU/mL) and placed in shaking incubator for 30 minutes at 3720 C. [0166] 3.15 mL samples created using 12 mL culture and a mixture of PBS buffer and sample compounds. [0167] 4. Placed under LED lamps for 2 hours (40 W/m.sup.2). [0168] 5. Samples diluted to 10.sup.4 and culture controls diluted to 10.sup.5 for plating through series dilutions. [0169] 6. 0.1 mL plated of each sample in triplicates on TSA base (10.sup.3 and 10.sup.4 for samples. 10.sup.4 and 10.sup.5 for controls). [0170] 7. Incubated overnight at 37 C. [0171] 8. Colonies counted next morning and recorded.