WO2005079569A1 - Compositions and methods for preserving plant material - Google Patents

Abstract

A composition nanocrystalline silver is used to preserve plant material. This prevents the growth of microorganisms, the formation of a biofilm, and thus extends the shelf life of plant material.

Description

Compositions and Methods for Preserving Plant Material

I. Field of Invention

[0001] This invention relates to compositions and methods for treating plant materials, and more particularly to preserving and/or extending the vase life of cut flowers. The compositions and methods also involve treating and/or preventing microbial contamination of plants, and to treating and preventing microbial plant diseases associated with biofilms.

II. Background of the Invention

[0002] As the expanding market for cut flowers grows, it has become increasingly important to extend the life of cut flowers. Plant stresses can induce the production of ethylene (the "death" hormone), which accelerates senescence, affects flowering, fruit setting and ripening, seed germination, and defense against pathogens among other plant functions. (1) This combination of factors, along with increasing consumption of cut and potted flowers and the current level of international trade of flowers, has created a need for novel and effective approaches to preserve the freshness and quality of cut flowers.

[0003] The literature describes commercial formulations that purport to extend the vase-life of flowers, such as roses, gerbera, carnation, sunflowers, chrysanthemum, gladiolas, among others (2, 3). These formulations usually include a sugar, which provides an energy source for the cut flowers and seems to protect against initial water loss (4), and may contain one or more microbial inhibitor(s) (5, 6, 7, 8). [0004] Currently, there are many methods to increase the shelf or vase life of cut flowers. Many of these methods are costly, ineffective, and inconsistent. Presently, floral preservatives mainly consist of sucrose and other sugars; germicides or biocides as disinfectants; and acids to adjust the pH of the water to 3.5 - 5.0. Common floral preservatives brands are Aquaplus, Floralife, Rogard/Silgard and Chrysal, Oasis and Vita Flora.

[0005] Alternative methods for floral preservation may include the following: conditioning or hydrating solutions which consist of water, a germicide, acidifier, and a wetting agent, which is used to restore dry-packed flowers that have wilted; impregnating solutions for protecting the plant material from the blockage of water by microorganisms; and loading or shipping compositions for extending the life of cut flowers that are shipped long distances. This method uses a higher concentration of sugar, but since sugar is also a primary source for the growth of microorganisms this treatment is not always effective.

[0006] It has been demonstrated that high microorganism count can cause premature wilting and senescence. (31 , 32, 33, 34) and thereby impact longevity of cut flowers. Microorganisms associated with cut flower premature senescence in vase solution can not only cause wilting by physically plugging the xylem (35), but they may also produce harmful metabolites that may accelerate flower death (36, 37). In addition, the production of exopolysaccharides by vase water bacteria aggravates xylem blockage, leading to diminished water uptake and early wilting. (38). Many microorganisms, including Erwinia spp. also produce ethylene that contributes to senescence in the plant.

[0007] There is also a need for a novel class of compounds with efficacy against miscrorganisms growing as biofilms. Biofilms are structured communities of microorganisms that exist attached to surfaces (microbial slime) (40). This is in constrast to the traditional view of microrgansims as floating single celled organisms (planktonics). Biofilms are the predominant form of growth in nature and very difficult to control, because they are less susceptible to antibiotics and biocides. (40). [0008] Biofilm problems in plants include vascular diseases (41 , 42), produce spoilage (43), seed contaminations, and shortened vase life of cut flowers. The presence of biofilms blocking cut flower water vessels has been indirectly described. It has been reported that cut surface of roses were covered by bacteria within two days after flowers were placed in water, also including the presence of bacterial "slime" (one of the hallmarks of biofilms). Clusters of bacteria and fungal hyphae were found inside the xylem vessels. (44). The report of yeast contamination reducing the vase-life of various cut flowers, which could not be removed even by repeated re-cutting of the stems (31), indicate the likeliness of a biofilm in the upper parts of water vessels. Biofilm clogging of xylem prevents water movement and induces water stress, leading to wilting, ethylene production and premature senescence. [0009] Microbial biofilms can grow on plant stems, leaves, roots, flowers, fruits and seeds. In addition, proper decontamination of surfaces is important for maximizing the quality of cut flowers and ornamentals, seeds and produce. Many currently available antimicrobial agents used for this purpose are not effective or not fully effective against biofilms, as they were designed and tested against organisms in the planktonic mode of growth. (43). Most biocides that have been developed are not as effective against biofilms. (Ceri et al., 1999). An effective treatment in removing or degrading biofilms in plant material needs to be developed.

[0010] There is still a need for an efficacious, inexpensive anti-biofilm agent with the following properties: sustained release of anti-biofilm agent, ability to remove or degrade biofilms, and a low level of toxicity. This would be extremely beneficial to a very perishable commodity by lowering costs, increasing quality of plant material, increasing customer satisfaction, and promoting industry growth.

III. Summary of the Invention

[0011] A need still exists for extending or improving the life of a flower after it has been cut or harvested. The inventors believe that one or more biofilms are present in the stem of a cut flower, and their presence in the xylem vessel lumen and/or interstitial portions of the stem prevents the effective use of agents that might prolong the life and quality of the cut flower. Biofilms are complex communities of microorganism that attached to surfaces. Biofilms may block the flow of water and nutrients to the flower through the xylem tubes and decrease the life and quality of the plant material. [0012] The invention describes a more efficient, practical and environmentally friendly way to maximize longevity and quality of plant cut floricultural products. The efficacy of inorganic and organic soluble salts of silver as antimicrobials has been demonstrated, but it has also been shown that they do not afford prolonged protection due to loss through removal or complexation of the free silver ions (45). A new alternative is available for silver-based antimicrobials, nanocrystalline silver compounds (46-49), which has been applied successfully in the medical field (50, 51 , 52). Nanocrystalline silver is superior to silver soluble salts because allows the sustained release of silver ions at antimicrobial active levels (45), and enhanced antimicrobial efficacy when compared to other silver compounds such as silver nitrate and silver sulfadiazine (48).

[0013] This invention demonstrates that stable, slow release nanocrystaline silver compounds, can be used as antimicrobials against bacteria and fungi pathogens, including biofilms, growing on plant surfaces.

IV. Detailed Description of the Invention

[0014] The present invention comprises compositions and methods for preserving the life or quality of plant material by contacting it with a composition comprising one or more preservative agents. The preservative agent comprises nanocrystalline silver. The invention may further include one or more anti-ethylene agents and/or one or more other additives selected from the group consisting of a nutrient, a pH adjuster or buffer, a source of carbon, a source of nitrogen, and/or a wetting agent.

[0015] In some embodiments of the invention, the compositions and methods may be used to preserve plants or parts thereof, preferably cut flowers.

[0016] In some embodiments of the invention, the compositions and methods may be used to treat or prevent one or more biofilms, and/or to treat or prevent one of more diseases.

[0017] The invention also comprises contacting the plant material with a composition comprising one of more preservative agents, thereby extending storage life or preserving the plant material, such as a cut flower.

[0018] In some embodiments of the invention, the nanocrystalline silver containing preservative may be used with other ingredients, including but not limited to a buffer or acidifiers (e.g., citric acid); nutrients, such as a source or nitrogen, a source of carbon, and/or a source of a sugar (e.g. glucose); ethylene inhibitors (e.g. hydroxyquinoline); or other biocides.

[0019] In some embodiments of the invention, the compositions and methods may include applying the preservative agent to any portion of a plant, including but not limited to a cut or wounded surface of a plant; the roots, stems, leaves, or the seeds. [0020] In accordance with some embodiments of the invention, any method of contacting the plant or portion thereof with a preservative agent may be used. Typical mechanisms for contacting the plant include but are not limited to spraying, immersing, and diffusing. Further, any storage or transport container can be impregnated with a preservative agent of the present invention so that the preservative agent comes into contact with a plant or a portion thereof. Typical containers include but are not limited to a bucket, carton, paper wrapper, plastic wrapper, vase, or box. One skilled in the art will readily recognize that a wide assortment of containers can and do come in contact with a plant; any of these containers can be used to facilitate contacting the plant of portion thereof with a preservative agent of the present invention. [0021] The compositions of the present invention may be used to treat a plant or portion thereof to eliminate or reduce one or more undesirable and/or deleterious microorganisms. The compositions of the present invention may be used to prevent one or more undesirable or deleterious microorganism from infecting a plant or portion thereof. In these embodiments of the invention, the preservative compositions and methods may be an anti- icrobial agent

[0022] The compositions of the present invention may be used to treat a plant or portion thereof to eliminate or reduce one or more undesirable and/or deleterious biofilms. The compositions of the present invention may be used to prevent one or more undesirable or deleterious biofilms from infecting a plant or portion thereof. In these embodiments of the invention, the preservative compositions and methods may be an anti-biofilm agent.

[0023] An embodiment of the invention comprises contacting plant material with - a preservative agent composition comprising a metal or metal compound, preferably nanocrystalline silver. One embodiment of the present invention comprises a composition comprising nanocrystalline silver, and its use for preserving and extending the shelf life of cut flowers.

[0024] An embodiment of the invention includes treating a plant or portion thereof by contacting it with a composition comprising a preservative agent, an ethylene inhibitor, or combinations thereof. In these embodiments of the invention, the compositions and methods preserve or extend the life of a plant or portion thereof, act as an anti-biofilm agent, and/or act as an antimicrobial agent.

[0025] Another further embodiment of the invention is contacting the plant material with the anti-biofilm agent by using proportion system. The anti-biofilm agent is mixed with water using a hose and water spigot which dispense the solution in the proper quantities automatically into a storage or holding container.

[0026] The invention may also include an apparatus and method for determining one or more anti-biofilm agents that are effective against one or more biofilms in plant material.

[0027] The preservative of the present invention comprises at least one metal or metal compound. In preferred embodiments of the invention, the preservative agent comprises nanocrystalline silver (e.g., N-Ag). Nanocrystalline silver, its formation, and its incorporation into a variety of compositions, can be found by reference to one or more of the following U.S. Patents: 6,723,350; 6,719,987; 6,692,773; 6,605,751 ;

6,238,686; 6,017,553; 5,985,308; 5,958,440; ; 5,837,275; 5,753,251 ; 5,681 ,575;

5,454,886; 6,516,633; and 6,017,390. All of these patents are herein incorporated by reference.

[0028] The metal or metal compound of the present invention relates to the use of one or more noble metals selected from silver, gold, platinum, and palladium but most preferably silver, in a nanocrystalline form, for the treatment and preservation of plant material. Among the noble metals, silver is preferred for such treatment. The nanocrystalline noble metal of choice may be used in the form of a nanocrystalline coating of one or more noble metals, a nanocrystalline powder of one or more noble metals, or a solution containing dissolved species from a nanocrystalline powder or coating of one or more noble metals. Nanocrystalline, as used herein typically refers to a grain size which is less than 100 nm in at least one dimension. See, for example,

U.S. Patent 6,692,773. Preferably, these noble metals are formed with atomic disorder, such that ions, clusters, atoms or molecules of the metals are released on a sustainable basis.

[0029] The nanocrystalline forms of these noble metals may be used in any of the following formats: i) nanocrystalline coatings of the noble metals on medical grade substrates, for example, dressings, fibers, and materials composed of for example polyethylene, high density polyethylene, polyvinylchloride, latex, silicone, cotton, rayon, polyester, nylon, cellulose, acetate, carboxymethylcellulose, alginate, chitin, chitosan and hydrofibers; ii) gels , formulated with nanocrystalline powders of the noble metals with such materials as carboxymethylcellulose , alginate , chitin, chitosan and hydrofibers , together with such ingredients as pectin and viscosity enhancers ; iii) creams , lotions , pastes and ointments formulated with nanocrystalline powders of the noble metals , for example as emulsions or with drying emollients ; iv) liquids , formulated as solutions by- dissolving nanocrystalline coatings or powders of the noble metals, for example as topical solutions, aerosols or mists ; v) powders, prepared as nanocrystalline powders of the noble metals , or as nanocrystalline coatings of the noble metals on biocompatible substrates in powder form, preferably on bioabsorbable and/or hygroscopic substrates such as : Synthetic Bioabsorbable Polymers : for example polyesters/polyactones such as polymers of polyglycolic acid, glycolide, lactic acid, lactide, dioxanone, trimethylene carbonate etc . , poly anhydrides , polyesteramides , polyortheoesters , polyphosphazenes , and copolymers of these and related polymers or monomers ; Naturally Derived Polymers : Proteins : albumin, fibrin, collagen, elastin; Polysaccharides : chitosan, alginates , hyaluronic acid; and Biosynthetic Polyesters : 3 -hydroxybutyrate polymers . [0030] Nanocrystalline powders (i.e., powders formed from particulates having nanocrystalline grain size) of one or more noble metals are most preferably prepared with atomic disorder by the procedures set out in WO 93/23092 and WO 95/13704, or as otherwise known in the art. The powders may be prepared as pure metals, metal alloys or compounds such as metal oxides or metal salts, by vapour deposition, mechanical working, or compressing in order to impart atomic disorder, as set out below, and as in the above-mentioned patent application. Mechanically imparted disorder is conducted by milling, grinding, hammering, mortar and pestle or compressing, under conditions of low temperature (i.e., temperatures less than the temperature of recrystallization of the material) to ensure that annealing or recrystallization does not take place. Alternatively, nanocrystalline powders may be prepared by preparing nanocrystalline coatings by physical vapour deposition to include atomic disorder in the manner set out above, onto a substrate such as a cold finger or a silicon wafer (or larger substrates), and then scraping off the coating to form a powder. A still further alternative method of powder preparation is to prepare nanocrystalline coatings, such as by physical vapour deposition to include atomic disorder as set out above, onto powdered substrates which are biocompatible. Particularly preferred substrates are bioabsorbable and/or hygroscopic powders such as chitin. [0031] Most preferably, powders of the present invention are sized at less than

100 .mu.m, and more preferably less than 40 .mu.m.

[0032] The prepared nanocrystalline powders may then be incorporated into or onto formulations by methods known in the art. For example, the powders may be layered onto the substrates (matrices or powders), mechanically mixed within the fibers of the matrix, impregnated into the matrix, by for example, physical blowing, or added to topical pharmaceutically acceptable composition ingredients. [0033] The anti-proliferative effects of the nanocrystalline powder may be achieved when the powder is brought into contact with an alcohol or a water-based electrolyte, thus releasing the noble metal ions, atoms, molecules or clusters. [0034] Nanocrystalline powders may be sterilized as described above, or may be prepared as preserved materials with known preservatives such as methyl paraben or propyl paraben. Alternatively, given the anti-microbial activity of the nanocrystalline powders themselves, they may be considered as being in a preserved form without the addition of preservatives.

[0035] Typically, the nanocrystalline noble metals will be formulated from the active ingredient, namely nanocrystalline powders or coatings of the noble metals, or dissolved species from such powders or coatings, in the form of: topical pharmaceutical compositions such as gels , pastes, ointments , creams , lotions, emulsions , suspensions or powders; or liquid pharmaceutical compositions prepared by dissolving nanocrystalline coatings or powders of the noble metals in pharmaceutically acceptable carriers such as water, for application in drop, mist or aerosol forms.

[0036] In the pharmaceutical compositions, the amount of the nanocrystalline metal powder may range broadly from about 0.001% to about 30% by weight, but will more preferably fall in the range of from about 0.01 to 5% by weight. Coatings of the nanocrystalline noble metals may be very thin, or thicker, depending on the desired duration of application. Typical coating thicknesses are in the range of 150 to 3000 nm thick. As liquid formulations, the amount of dissolved noble metal will typically range between about 0.001 to 1% by weight.

[0037] Nanocrystalline gels may be formed from the nanocrystalline metal powder in admixture with gelling agents such as carboxymethyl cellulose (CMC), polyvinyl alcohol (PVA), collagen, pectin, gelatin, agarose, chitin, chitosan, and alginate, with the gelling agent comprising between about 0.01-20% w/v. (0038] Dosage forms for the topical administration of compositions of the nanocrystalline noble metals include various mixtures and combinations. Examples include sprays, mists, aerosols, lotions, creams, solutions, gels, ointments, pastes, emulsions, and suspensions. The active compound can be mixed under sterile conditions with an acceptable carrier, and with any preservatives, buffers, or propellants which may be required. Topical preparations can be prepared by combining the noble metal powder with conventional acceptable diluents and carriers commonly used in topical dry, liquid, cream and aerosol formulations. Ointment and creams can, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. An exemplary base is water. Thickening agents which can be used according to the nature of the base include aluminum stearate, hydrogenated lanolin, and the like. Lotions can be formulated with an aqueous or oily base and will, in general, also include one or more of the following: stabilizing agents, emulsifying agents, dispersing agents, suspending agents, thickening agents, coloring agents, perfumes, and the like. Powders can be formed with the aid of any suitable powder base, e.g., talc, lactose starch and the Jike. Drops can be formulated with an aqueous base or non-aqueous base, and can also include one or more dispersing agents, suspending agents, solubilizing agents, and the like. [0039] Multiple inactive ingredients are generally incorporated in the formulations to improve cosmetic acceptability, and are optional ingredients in the formulations . Examples of ingredients are emulsifiers , thickening agents , solvents, ant i -foaming agents , preservatives, fragrances , coloring agents , emollients , and fillers . [0040] In preferred embodiments of the invention, the preservative is incorporated into a matrix or the like. Any form of matrix may be suitable for use in the practice of this invention. An exemplary matrix and the method of forming a matrix that includes nanocrystalline metal including silver are disclosed in U.S. Patent 6,605,751 , and U.S. Patent 5,985,308, incorporated herein by reference. [0041] A preferred embodiment of the present invention comprises a hydratable matrix material that has an antimicrobial agent, such as a heavy metal, most preferably, silver, incorporated into the matrix. The matrix preferably also comprises components that stabilize and control the release of the active agent into the surrounding environment when used.

[0042] In a preferred embodiment of the present invention, active agents are incorporated directly, or may be incorporated by sequentially adding components or precursors of the active agent to the matrix of the devices, and having the precursors of the active agent in or on the matrix. The agents may be incorporated by absorption or adsorption of agents or precursors by the matrix, and preferably by incorporation during the polymerization of the matrix. It is theorized that the release of the active agents may be controlled via manipulation of concentration parameters, movement of water through the matrix and the degree of cross-linking in the matrix. In another preferred embodiment, the wound dressings comprise a stranded configuration, wherein the strands extend from at least one common region and the strands themselves comprise a polymer matrix.

[0043] Other forms of the matrices of the present invention, such as molded articles, are also contemplated by the present invention. Other forms also include films, sheets, fibers and amorphous gels. The matrices of the present invention can be dipped or applied in methods known to those skilled in the art to articles or devices. [0044] The present invention may also include the incorporation and stabilization of silver onto and within the hydrophilic fibers of cross-linked and non-cross-linked celluloses such as carboxymethyl cellulose and hydroxymethyl cellulose, cotton, rayon, and of fibers made from polyacrylates and other synthetic and natural polymers, and fibers of calcium alginates that may be used as a primary contact sustained-release silver antimicrobial materials.

[0045] The preservative agents incorporated into the matrices and devices of the present invention may be used for a variety of applications where there is a need for the presence of the active agent. A particularly preferred use is in the treatment and preservation of plant materials, particularly cut flowers. The composition may also include antimicrobial agents, including but not limited to antifungal agents, antibacterial agents, anti-viral agents and anti-parasitic agents, growth factors, angiogenic factors, anaesthetics, mucopolysaccharides, and metals.

[0046] Examples of antimicrobial agents that can be used in the present invention include, but are not limited to, isoniazid, ethambutol, pyrazinamnide, streptomycin, clofazimine, rifabutin, fluoroquinolones, ofloxacin, sparfloxacin, rifampin, azithromycin, clarithromycin, dapsone, tetracycline, erythromycin, ciprofloxacin, doxycycline, ampicillin, amphotericin B, ketoconazole, fluconazole, pyrimethamine, sulfadiazine, clindamycin, lincomycin, pentamidine, atovaquone, paromomycin, diclazarii, acyclovir, trifluorouridine, foscamet, penicillin, gentamicin, ganciclovir, iatroconazole, miconazole, Zn-pyrithione, heavy metals including, but not limited to, gold, platinum, silver, zinc and copper, and their combined forms including, salts, such as chloride, bromide, iodide and periodate, and complexes with carriers, and other forms.

[0047] Preferably, the hydrophilic matrix material is constructed from a natural or synthetic polymer and a non-gellable polysaccharide. Natural hydrophilic polymers that may be used include, but are not limited to collagen, arnal hide, hyaluronic acid, dextran and alginate. Additionally included are hydrophilic fibers of cross-linked and non-cross- linked celluloses such as carboxymethyl cellulose and hydroxymethyl cellulose; cotton, rayon, and of fibers made from polyacrylates; and fibers of calcium alginates that may be used as a primary contact sustained release silver antimicrobial material. Synthetic polymers that may be used include, but are not limited to polyacrylamide, polyvinyl's (PVP, and PVC), polyacrylate, polybuterate, polyurethane foam, silicone elastomer, rubber, nylon, vinyl or cross linked dextran. If cross-linked dextran is used, it is preferred that the molecular weight of the dextran polymer is between 50,000 and 500,000. The most preferable non-gellable polysaccharide is a non-gellable galactomannan macromolecule such a guar gum. A range of guar gum between approximately 0.01 kg to 100 kg, preferably between approximately 0.1 kg to 10 kg, and most preferably between approximately 0.5 kg to 2 kg is generally sufficient. Other non-gellable polysaccharides may include lucerne, fenugreek, honey locust bean gum, white clover bean gum and carob locust bean gum.

[0048] The matrix of the preferred embodiment preferably comprises polymerized chains of acrylamide monomer, wherein the acrylamide monomers are cross-linked with a cross-linking agent, a non-gellable polysaccharide, and an active agent or pharmaceutical directly encapsulated into micro-cavities therein. A range of acrylamide between approximately 1 kg to 100 kg, preferably between approximately 2 to 50 kg, and most preferably between approximately 5 kg to 20 kg is generally sufficient. A preferred matrix comprises a cross-linked polyacrylamide scaffolding that enmeshes guar gum as disclosed in U.S. Pat. No. 5,196,160 to Nangia, incorporated herein by reference.

[0049] In the present invention, nanocrystalline silver can be administered in the following formats: bound to a solid matrix, gels formulated with nanocrystalline silver powders, creams, lotions, pastes, ointments formulated with nanocrystalline silver powders; liquids formulated as solutions by suspension of nanocrystalline silver powders together with one or more, The preferred methods comprises formulating nanocrystalline silver as bound to a solid matrix or aqueous suspensions. [0050] In the present invention nanocrystalline silver may be formulated in the form of powders, topical compositions or liquid compositions. The amount of nanocystalline silver may range broadly from 1 to 200 for planktonic microorganisms and 10 to 5000 ppm for biofilm microorganisms, but preferably with nanoparticle size of no more than 1000 nanometers, depending on upon plant material. Nanosizing the silver ions lead to economical utilization and greater effectiveness. [0051] An embodiment of the invention includes assaying plant material for the presence of a biofilm and/or determining one or more anti-biofilm agents effective against the biofilm.

[0052] In accordance with the present invention, the effectiveness of a composition of the present invention may be determined by any process or means that involves contacting the biofilm with the active agent. An exemplary device and process is shown in U.S. Patent No. 6,052,423 and 6,326,190, each incorporated herein by reference.

[0053] In the present invention, the plant material may be assayed using any assay device for detecting a biofilm and or for determining an anti-biofilm agent. An exemplary embodiment of the invention comprises determining one or more anti-biofilm agents, preferably using a biofilm assay device. One method includes, but is not limited to, analyzing biofilms and their reaction to anti-biofilm agents from exemplary device and process shown f^"π U.S. Patent Nos. 6,051,423; 6,051,423; 6,326, 190; 6,410,256; 6,596,505; 6,599,696; and 6,599,714.

[0054] In the present invention, the preferred method is the Minimal Biofilm

Eradication Concentration (MBEC) technique which consists of growing identical 24 - hour biofilms on 96 pegs arrayed in 12 rows and 8 columns. The biofilms then are challenged with decreasing concentrations of selected antibiotics and/or biocides. After a certain challenge time (generally one hour), the biofilms are placed in 96 individual wells of growth media and ultra-sonicated for recovery of any surviving organisms. After culturing overnight, the wells are checked for turbidity. Clear, transparent wells indicate complete deactivation of the biofilm. Conversely, turbidity ("growth") indicates lack of complete deactivation of the biofilm. Where needed, the bacterial suspensions can be culture of agar plates, with or without 10-fold serial dilution, to estimate the surviving rate of the microorganisms in biofilms. Log reductions can be estimated to evaluate efficacy of biocide. In this method, the bacteria after incubated to form the biofilm is treated with anti-bacterial agent and removed from the adherent site for further incubation. The adherent sites are then treated with the anti-bacterial agent. The biofilm is analyzed using a device containing channels for flow of liquid growth mediums to test for sensitivity.

Definitions

[0055] As used herein, plant material refers to any plant or vegetable, or parts thereof, including flowers, fruits, produce, seeds, stems and wood. In preferred embodiments of the invention, cut flowers refer to flowers and stems that have been cut to be used alone or in combination with other flowers in a flower arrangement. [0056] As used herein, anti-biofilm agent refers to any element, chemical, biochemical, or the like that is effective against a biofilm. Typical anti-biofilm agents are those that have anti-microbial, anti-bacterial, anti-fungal or anti-algal properties. Metal and metal compounds, preferably nanocrystalline silver, have been shown generally to have anti-bacterial and ethylene inhibiting properties, and are preferred anti-biofilm agents in accordance with the present invention. The compound 8-hydroxyquinoline citrate has been shown generally to have ethylene inhibiting properties, and is the preferred ethylene inhibiting agent in accordance with the present invention. [0057] As used herein, preservatives or similar words include any element, chemical or biochemical or the like that can be used to preserve or extend the shelf like of a plant material, such as a cut flower. A preservative may be an anti-biofilm agent, or may be used in combination with an anti-biofilm agent, or may be used after an anti- biofilm agent is removed or degraded a biofilm.

[0058] "Sustained release" or "sustainable basis" are used to define release of atoms, molecules, ions or clusters of a noble metal that continues over time measured in hours or days, and thus distinguishes release of such metal species from the bulk metal, which release such species at a rate and concentration which is too low to be effective, and from highly soluble salts of noble metals such as silver nitrate, which releases silver ions virtually instantly, but not continuously, in contact with an alcohol or electrolyte.

[0059] "Therapeutically effective amount" is used herein to denote any amount of a formulation of the nanocrystalline noble metals which will exhibit an anti-proliferative effect when applied to a plant material. A single application of the formulations of the present invention may be sufficient, or the formulations may be applied repeatedly over a period of time, such as several times a day for a period of days or weeks. The amount of the active ingredient, e.g., the nanocrystalline noble metal in the form of a coating, powder or dissolved in liquid solution, will vary with the conditions being treated, the stage of advancement of the condition, and the type and concentration of the formulation being applied. Appropriate amounts in any given instance will be readily apparent to those skilled in the art or capable of determination by routine experimentation.

[0060] "Nanocrystalline" is used herein to denote single-phase or multi-phase polycrystals, the grain size of which is less than about 100, more preferably <50 and most preferably <25 nanometers in at least one dimension. The term, as applied to the crystallite or grain size in the crystal lattice of coatings, powders or flakes of the noble metals, is not meant to restrict the particle size of the materials when used in a powder form.

[0061] "Powder" is used herein to include particulate sizes of the nanocrystalline noble metals ranging from nanocrystalline powders to flakes.

[0062] Acidifier: Chemicals that lower the pH of a solution, making it more acid.

Common ones used in flower foods include citric acid and aluminum sulfate.

[0063] Anti-ethylene (chemical treatments): Agents that can delay ethylene induced senescence by inhibiting either the synthesis or action of ethylene.

[0064] Bent neck: Bending or drooping of flower heads, immediately below where they are attached to the stems. Biocide: Any chemical that can kill or reduce the levels of microorganisms.

[0065] Buckets (displav/storaqe/transport) : Any device that can hold cut flowers in solution, such as five-gallon buckets to stacking wet packs that are used to ship flowers long distances in solution and holding containers used for hydrating and displaying flowers.

[0066] Citric acid: Common acidifier often used for in flower food compositions and hydration solutions.

[0067] Cut flowers: Flowers marketed after removal from parent plants (ex: roses, gerbera). Also called fresh flowers. . [0068] Cut foliage: Branches and leaves used in flower arrangements and bouquets.

[0069] Flower food: Ingredients that can extend the life of cut flowers when added to the vase water. Also called flower preservative.

[0070] MIC: Minimal Inhibitory Concentration - minimal concentration of an antimicrobial that is effective to inhibit microbial growth.

[0071] MBC: Minimal Bactericidal Concentration - minimal concentration of an antimicrobial that can effectively eradicate planktonic population of microorganisms.

[0072] MBEC: Minimal Biofilm Eradication Concentration - minimal concentration of an antimicrobial that can effectively eradicate biofilms.

[0073] pJH : A measurement of the acidity of a solution. A pH of 7.0 is neutral.

Lower pH values are more acidic and above 7.0 they are alkaline.

[0074] Planktonic: Microorganisms growing as floating, single cells, which is part of their life cycle.

[0075] Post-harvest: The period from harvest on for crops such as cut flowers and potted plants, and vegetables.

[0076] Shattering : Falling off flowers and/or petals.

[0077] Sustained release: release of atoms, molecules, ions or clusters of an antimicrobial or noble metal that continues over time, measured in hours or days.

[0078] Vaselife: The life of flowers starting when they arrive at the final consumer.

[0079] Although a few preferred embodiments have been described, it will be appreciated by those skilled in the art that various changes and modifications might be made without departing from the scope of the invention. The terms and expressions in the preceding specification have been used therein as terms of description and not of limitation, and there is not intention in the use of such terms and expressions of excluding equivalents of the features shown and described or portions thereof, it being recognized as the scope of the invention as defined and limited only by the claims that follow.

Examples of embodiments of the invention The present invention will be further described in detail with reference to the following working examples. Note, however, that the present invention is not restricted to these examples.

EXAMPLE 1 : Evaluation of nanocrystalline silver efficacy against plant- associated -bacteria planktonic and biofilms in vitro (abiotic surface), and comparison with other biocides The objectives of this study were to evaluate nanocrystalline silver (N-Ag herein) in vitro as a potential antimicrobial against planktonic (floating and single-cells) and biofilm populations (attached to a surface and to each other) of bacteria associated to plant and seed diseases, and post-harvest decay, in comparison with other commonly used biocides. The study was carried out using the MBEC device, which permits in vitro modeling of microbial biofilms and high throughput technology for the rapid selection of antimicrobials specifically targeted against biofilms. (Ceri et al., 1999). The bacterial species tested include Erwinia carotovora Ecc26 (causes vascular diseases in potatores, soft rot of vegetables, and premature death of cut flowers), Pseudomonas syringae pv. phaseolicola HB-9 (causes seed- transmitted bean leaf spot), and Pseudomonas aeruginosa PA14 (is a multi-host pathogen) Biocides tested were one of the following: 1) Bleach (sodium hypochlorite), 2) N- Ag (from silver coated burn dressing, Acticoat, Nucryst), 3) Silver Nitrate or 4) 8- Hydroxy-quinoline sulfate (HQS). Bleach, Silver nitrate and 8-Hydroxyquinoline have been used as a component of plant preservatives. N-Ag was tested by placing pieces of Acticoat (Nucryst) bandage into sterile distlled water or by using water saturated with nanocrystalline silver, a solution that was prepared by immersing 1 sq. inch of Acticoat in 5mL of water for 6h at room temperature, which generates an equilibrated solution containing about 70 parts per million (ppm) of silver ions. Silver nitrate or HQS were solutions prepared weight/volume in sterile distilled water while a solution of bleach was prepared volume/volume to produce a starting testing concentration of 500 ppm. The results are summarized on Table 1. TABLE 1: In vitro efficacy of selected biocides against biofilms formed by Ecc26, Pa14 and PsyrHB9. Biocide Ecc26 PA14 PsHB9 Planktonic Biofilm Planktonic Biofilm Planktonic Biofilm (MBC- ppm) (MBEC-ppm) (MBC - ppm) (MBEC-ppm) (MBC - ppm) (MBEC-ppm) Bleach <16 <16 <16 <=16 <16 <16 HQS 500 > 500 250 n/a 125 500

Nano Ag strips < 0.25 cm² 2.0 cm² < 0.25 cm² 1.5 cm² < 0.25 cm² > 1.0 cm²

Nano Ag liquid 2 > 70 1 > 70 1 > 70 AgNOs <16 > 500 <: 8 > 500 < 8 > 500

MBC values (Minimal Bacteriocidal Concentration) are in parts per million and represent the minimal concentration of the antimicrobial agent able to kill all bacteria growing in planktonic mode. MBEC stands for Minimal Biofilm Eradication Concentration and represent the minimal concentration of antimicrobial required to kill all microorganisms growing in a biofilm. MBEC values for all microorganisms tested were higher than the MBC values, as expected due to the enhance resistance of biofilms to antimicrobials. The results demonstrated that N-Ag, either bound to Acticoat bandage strips or in the form of a saturated solution, showed more efficacy against the bacteria tested than HQS or silver nitrate. Eradication of planktonic populations was achieved at concentrations of 1 to 2 ppm as prepared by 2-fold serial-diluted from an equilibrated solution of N-Ag with starting concentration of (~ 70ppm), demonstrating the great efficacy of N-Ag for controlling microbial populations in solution. This characteristic is important for proper control of microorganisms growing in vase water, which prevents their association with plant tissue and consequent biofilm formation. In addition, N-Ag delivered into the treatment solution bound to a solid matrix, showed higher efficacy in eradicating biofilms that the saturated solutions of N-Ag prepared by soaking of Acticoat in water. The presence of the solid matrix warranted continuous release of silver ions into the system, while the solution saturaded with released, without the presence of a sustained-release mechanism, would become depleated of silver overtime. N-Ag was proven efficacious against microorganisms associated with both plant diseases and seed contamination microorganisms (Erwinia and P. syringae) and bacteria associated with cut flower premature death (Erwinia and Pseudomonas spp.) N-Ag delivered as bound to the Acticoat bandage also showed good efficacy against bacterial biofilms, measured according to the area of the silver-coated bandage, which affects the kinetics of silver ion release. Thus, this experiment demonstrated that N-Ag can be used at much lower concentrations that other commonly used biocides against both planktonic and biofilm populations of plant-associated bacteria. N-Ag can be used to prevent or treat biofilms associated with plant material.

EXAMPLE 2 Evaluation of nanocrystalline silver efficacy against plant-associated -bacteria planktonic and biofilms in vitro (wooden surface) Balsa wood sticks were also used as a complementary technique for for biofilm formation and evaluation of anti-biofilm activity of Acticoat N-Ag. Wooden surfaces mimic the porous nature of plant vascular tissue (xylem is basically "dead" wood) and also the plant vascular tissue chemical composition (Marques et al., 2002). Erwinia carotovora Ecc26 was the model strain for these tests and for further in vivo studies, for it is a species commonly associated with flower decay and other plant diseases. Sterilized balsa wood cuts measuring 2.0 cm in long x 0.6 cm wide x 0.2 cm thick were added to bacterial suspensions incubated under orbital shaking for 8h. The incubation time was defined based on previous growth curves indicating that, in average, Erwinia populations reached 7.74 log for planktonics in liquid culture and biofilms recovered from wood cuts produced cell counts of 7.96 log. After incubation, wood cuts were rinsed in phosphate buffer and transferred into a 12-well plate containing 1.0 and 2.0 square inch pieces of Acticoat bandage, immersed in 1.5 mL of sterile distilled water, in triplicate. Bacterial suspension containing planktonic cells were transferred (0.1 mL) into 12-well plate wells containing 0.25 and 1.0 square inch pieces of Acticoat bandage, immersed in 1.5 mL of sterile distilled water, also in triplicate. Control treatments containing only sterile water were run side-by-side for both biofilms and planktonic populations. After incubation for 16h, planktonic cells were serial diluted in phosphate buffer (10-fold) and 0.02 ml of each dilution was plated on agar. Colonies were counted after 24h, and cell counts presented are based on number of colony forming units (cfu). The wood pieces containing treated biofilms were rinsed in phosphate buffer and were individually transferred to 50mL screwcap conical tubes containing 10 mL of phosphate buffer. Wood cuts were ground using a tissue homogenizer in 3 pulses of 1 minute. Another 3.5 mL of phosphate buffer and 1.5 mL of a 1 % solution of tween 20 were added to the suspension containing the ground wood and biofilm, bringing the total volume of suspension to 15.0 mL and the final concentration of tween 20 to 0.1%. This was followed by vigorous vortexing of wood debris suspension for 2 min., followed by 30 min. of sonication to ensure maximum recovery of biofilm from the wood surfaces. Finally, recovered biofilm suspension was serial diluted and plated as described for planktonics, As shown in Table 2, N-Ag was ver efficacious as a biocide for plant pathogenic bacteria growing both in planktonic mode, and as complex biofilms associated with wood tissue. The addition of 1.0 square inch of Acticoat was enough to completely erradicate planktonic populations in 16h with a 8.78 log reduction. The same conditions against biofilm caused only amodest 1.74 log reduction in biofilm population, which was expected, as biofilms have an innate higher resistant to anti-microbials. However, a drastic reduction of the biofilm population (6.62 Log) was achieved with a 2.0 square inch piece of Acticoat, and minor dosage adjustments can warrant complete eradication of biofilm population.

Table 2. Treatment of biofilms formed on wooden surfaces and planktonic populations grown in presence of wood sticks with Acticoat bandages for 16h

Treatment Planktonic Biofilm Log reduction Log Reduction Planktonic Biofilms

Control no N-Ag 8.78 8.27

0.25 inch² piece of Acticoat 1.06 Not tested- 7.72

1.0 inch² piece of Acticoat 0.00 6.53 8.78 1.74

2.0 inch² piece of Acticoat Not tested 1.65 6.62 The high efficacy presented against complex biofilms associated with plant vascular tissue demonstrated in this assay reveal the potential of application of nanocrystalline silver-based compositions for the treatment of plant biofilm diseases. This application could be particularly useful in the treatment of plant vascular diseases, such as: soft rot and blackleg of potatoes caused by Erwinia carotovora (subspecies carotovora and atroseptica, respectively) in the field; ring rot of potatoes caused by Clavibacter michiganensis subsp. sepedonicus (ring rot of potatoes), Xylella fastidiosa vascular diseases such as Pierce's disease of grapes, citrus variegated chlorosis in sweet oranges, alfalfa dwarf, almond leaf scorch, among others, and Ralstonia solenacearum induced diseases in various crops. All these diseases are currently untreatable. Thus, examples 1 and 2 combined demonstrate that N-Ag is very efficacious against planktonic populations, and against biofilms formed by plant pathogenic bacteria on both inert, solid surfaces (example 1) and porous, natural surfaces (example 2), which indicate strong potential as a novel compound to be used on control, prevention and treatment of plant biofilm infections.

EXAMPLE 3 Evaluation of nanocrystalline silver as an enhancer of cut flower longevity in in vivo trials Fresh premium wedding white roses produced in Alberta were treated with bleach,

8-hydroxyquinoline and nanocrystaline silver, in comparison to flowers placed in tap water without any additive. Upon receiving, flower stems were cut under water and roses were placed individually in long glass culture tubes containing 25 mL of treatment solution, 5 flowers /treatment, at room temperature. The tubes were not sterilized, neither were the tap water or biocide solutions, to mimick the "real life" conditions of "in site" future use by retailers or consumers. Treatments were: A) Tap water (negative control); B) Tap water inoculated with soft rot bacteria (Erwinia carotovora subps. carotovora = Ecc hereafter); C) Tap water and bleach (0.1%); D) Tap water and bleach (0.1%) inoculated with soft rot bacteria; E) Tap water and N-Ag (1 square inch fragment of Acticoat burn dressing per tube); F) Tap water and N-Ag (1 square inch fragment of Acticoat burn dressing per tube), inoculated with soft rot bacteria; G) Tap water and 8- Hydroxyquinoline sulfate (HQS) (0.2g/L) H) Tap water and 8-Hydroxyquinoline sulfate (HQS) (0.2g/L), inoculated with soft rot bacteria. The longevity of the flower was determined based upon several characteristics, such as: retention of flower color, damage to plant tissues (flower parts, leaves, stems), firm pedicels (bent neck), leaf and petal wilting and shattering, bud opening, overall correlating it to determine the vase life. (Salinger, Acta Hort. 41 ). After 4 days of treatment, the inoculated control sample containing tap water and bacteria (B), already presented dead flowers. The inoculated control (B) compared poorly with the N-Ag and HQS-treated inoculated flowers (F and H). Flowers were treated with bleach failed to completely open, their petals presented marginal browning and leaves showed spotting, indicating that bleach seems to be toxic to the flowers at the concentrations used. After 8 days of treatment, N-Ag was clearly superior to all other treatments as a flower preservative and anti-microbial under heavy microbial exposure (inoculated samples) and also worked really well in maintaining the quality and shape of non- inoculated flowers, better than hydroxyquinoline. At the end of the treatment, after 10 days, flowers treated with nanocrystalline silver outlasted the ones in all other treatments by at least 2 days. The longevity data based are presented in tables 3 and 4 (average of 5 flowers).

Table 3. Average longevity of cut roses under treatments without bacterial challenge Treatment Longevity Increase in longevity Water 6.2 days Water and Silver 8.2 days 32.3 % Water and Bleach 5.7 days No gain Water and HQC 7.0 days 13.0%

Table 4. Average longevity cut roses under treatments with bacterial challenge Treatment Longevity Increase in longevity

Water with Ecc 3.8 days

Water/Ecc and Silver 7.4 days 94.7%

Water/Ecc and Bleach 5.7 days 50.0%

Water/Ecc and HQC 5.6 days 47.4%

The results set forth in Table 3 demonstrate that the roses placed in tap water and bleach had the shortest life lifetime. The use of 8-hydroxyquinoline enhanced vase life in average by about 13%. The use of 1.0 sq. inch of Acticoat bandage providing continuous release of N-Ag increased flower life by 32 %. When flowers where challenged by the presence of a soft rot bacteria, the results were even more dramatic. While untreated flowers lasted 3.8 days in average, bleach and HQS treated flowers showed an increase of 50% and 47% in vase life, respectively. However, flowers treated with bleach were very poor in quality in comparison to HQS. The use of 1.0 sq. inch Acticoat bandage coated with N-Ag approximately doubled the rose lifetime, with a vase life increase of nearly 95%. The results of this example clearly indicate that the use of Nanocrystalline silver, delivered in the form of Acticoat bandages had a beneficial effect on fresh flower lifetime and quality, and was superior to a commonly used household preservative bleach and a commercially recommended preservative, HQS. These observations on longevity were correlated to microbial counts of the vase water, in tests performed as follows. Vase water samples were collected out of three tubes per treatment, at various time points, serial diluted up to 1000 times and 0.1 mL was spread plated on Nutrient Agar (NA). Plates were incubated at room temperature and colonies were counted after 24h-48h. The microbial counts correlated with the longevity and quality of flowers, i.e., vase water presenting lower microbial counts showed the best overall results.

Table 5. Microbial counts of flower vase water for all treatments over time (numbers in Iog10). Treatment Day 2 Day 4 Day 6 Da δ

No microbial challenge

A-Tap water 4.70 5.77 7.0 8.0

C-Tap water/bleach 0.0 4.38 7.0 8.0

E-Tap water/N-Ag 1.0 0.0 0.0 0.48 2.46

G-Tap water/HQS 0.49 4.20 4.58 8.0

Microbial challenge

B-Tap water/Ecc 7.0 7.0 8.0 8.0

D-Tap water/bleach/Ecc 0.0 5.39 7.0 8.0

F-Tap water/N-Ag 1.0/Ecc 1.81 2.97 3.53 5.28

H-Tap water/HQS/Ecc - 4.23 3.62 7.56

Again, based on microbial counts, N-Ag outperformed bleach and hydroxyquinoline as an anti-microbial and flower preservative in vivo. In treatments where no bacteria was inoculated, at the first sampling 2 days after treatment, bleach and HQS showed similar anti-microbial activity to N-Ag. However, at day 4 the differences on microbial counts between treatments were striking. Tap water with no bacteria added presented microbial counts of 5.77 log, in comparison to 4.38 log on bleach treated water and 4.2 log in HQS treated water. By contrast, nanocrystalline silver treatments still had no microbial growth at all at this point. After 8 days, all treatments had microbial populations in the 8 log range, while N-Ag treatment had a much lower microbial count (over 5.5 log reduction in relation to any other treatment), 2.46 log. The flower treatment challenged with bacterial inoculum showed the same tendency, with an overall superior performance of N-Ag over the other treatments. The microbial counts correlated with the longevity and quality of flowers, i.e., vase water presenting lower microbial counts showed the best overall results. The initial good efficacy of bleach and HQC was not long lasting, as they get used or degraded overtime. The continuous release of silver ions by N-Ag in aquous solutions is a clear benefit for the use of this compound as a flower preservative, as there will be no need to change the vase water and flower food daily. EXAMPLE 4 Evaluation of nanocrystalline silver as a enhancer of cut flower longevity of roses in vivo trials using flower food composition including sugar and acidifier agent The objective of the study was to evaluate the efficacy of nanocrystalline silver when combined to add to conventional components of plant preservative solutions: 1) water, sugar and citric acid; 2) water, sugar, citric acid and 8-hydroxyquinoline. Flower treatments for this trial were as follows: A) Tap water and 2% sucrose (weight/volume) and citric acid to pH 4.5 (referred herein as WSC). B) WSC solution inoculated with soft rot bacteria (Erwinia carotovora subps. carotovora = Ecc hereafter); C) WSC solution and N-Ag (0.5 square inch fragment of Acticoat burn dressing per tube); D) WSC solution and N-Ag (0.5 square inch fragment of Acticoat burn dressing per tube), inoculated with soft rot bacteria; E) WSC solution and N-Ag (1.0 square inch fragment of Acticoat burn dressing per tube); F) WSC solution and N-Ag (1.0 square inch fragment of Acticoat burn dressing per tube), inoculated with soft rot bacteria; G) WSC solution and 8-Hydroxyquinoline sulfate (HQS) (0.2g/L) H) WSC solution and 8- Hydroxyquinoline sulfate (HQS) (0.2g/L), inoculated with soft rot bacteria. Yellow roses were obtained from Growers Direct, Calgary, AB, and each treatment contained 5 flowers. For this trial, in addition the use of flower food components in the treatment, the bacterial challenge was started 24h after flowers were placed in the vase solution. Again, the best results in these trials were achieved with N-Ag, in comparison to untreated control or hydroxyquinoline. The use of N-Ag as a preservative nearly doubled the longevity of roses under microbial challenge and increased the vase life of roses in tap water and flower solution by over 50%. (Table 6 and 7).

Table 6. Average longevity cut roses under flower food solution treatments without bacterial challenge Treatment Longevity Increase in longevity

Water/sugar/citric ac. 5.2 days

Water/sugar/citric ac. + N-Ag 0.5 6.6 days 27.0 % Water/sugar/citric ac. + N-Ag 1.0 8.0 days 53.8 % Water/sugar/citric ac. + HQS 6.2 days 19.2 %

Table 7. Average longevity cut roses under flower food solution treatments with bacterial challenge Treatment Longevity Increase in longevity

Water/sugar/citric ac. 3.5 days Water/sugar/citric ac. + N-Ag 0.5 6.8 days 94% Water/sugar/citric ac. + N-Ag 1.0 6.6 days 88% Water/sucrose/citric ac. + HQS 5.4 days 54%

N-Ag also showed a much superior anti-microbial efficacy than hydroxyquinoline (Table 8), which correlates to the longevity data.

Table 8. Microbial counts of flower food solution treatments over time (numbers in Iog10).

Treatment Day 2 Day 4 Day 6 Day 8

No microbial challenge

A-Water/sugar/citric ac.(WSC) 6.86 6.50 6.62 7.32

C-WSC/N-Ag 0.5 0.00 0.51 1.63 2.19

E-WSC/N-Ag 1.0 0.69 0.55 0.93 2.42

H-WSC/HQS 5.80 7.48 7.27 7.76

Microbial challenge

B-WSC/Ecc 7.38 7.42 7.74 8.00

D-WSC/N-Ag 0.5/Ecc 0.92 3.57 4.44 4.71

F-WSC/N-Ag 1.0/Ecc 0.0 2.83 4.71 5.16

H-WSC/HQS/Ecc 5.94 7.72 7.73 7.64 EXAMPLE 5. Evaluation of nanocrystalline silver as a enhancer of cut flower longevity in flower food composition and evaluation of synergistic effect pf N-Ag and HQS The objective of the study was to investigate the performance of a combination of N-Ag and hydroxyquinoline in extending the life of cut flower and antimicrobial activity in comparison to either N-Ag or HQS alone. Flower treatments for this trial were as follows: A) Tap water and 2% sucrose (weight/volume) and citric acid to pH 4.5 (referred herein as "base solution"). B) WSC solution inoculated with soft rot bacteria (Erwinia carotovora subps. carotovora = Ecc hereafter); C) WSC solution and N-Ag (1.0 square inch fragment of Acticoat burn dressing per tube); D) WSC solution and N-Ag (1.0 square inch fragment of Acticoat burn dressing per tube), inoculated with soft rot bacteria; E) WSC solution and N-Ag (1.0 square inch fragment of Acticoat burn dressing per tube) and (HQS) (0.2g/L); F) WSC solution and N-Ag (1.0 square inch fragment of Acticoat burn dressing per tube), inoculated with soft rot bacteria (HQS) (0.2g/L); G) WSC solution and 8- Hydroxyquinoline sulfate (HQS) (0.2g/L); H) WSC solution and 8-Hydroxyquinoline sulfate (HQS) (0.2g/L), inoculated with soft rot bacteria. White roses were obtained from signle source, each treatment contained 5 flowers. Flowers were placed together in beakers containing each treatment solution. Again, flowers treated with N-Ag outlasted non-treated or HQS treated flowers (Table 9). In addition, a clear anti-rh.crobial synergistic effect between N-Ag and 8- Hydroxyquiniline sulfate has been demonstrated. Data are summarized on Tables 11 and 12.

Table 9. Average longevity cut roses under flower food solution treatments without bacterial challenge

Treatment Longevity Increase in Ion

Water/sugar/citric ac.(WSC) 5.6 days

WSC/N-Ag 1.0 8.4 days 50.0%

WSC/N-Ag 1.0/HQS 7.2 days 28.6%

WSC/HQS 6.6 days 17.8% Table 10. Average longevity cut roses under flower food solution treatments with bacterial challenge Treatment Longevity Increase in longevity

WSC/Ecc 3.6 days

WSC/N-Ag 1.0/Ecc 5.6 days 35.7%

WSC/N-Ag 1.0/HQS/Ecc 6.5 days 80.5%

WSC/HQS/Ecc 5.6 days 35.7%

Table 11. Microbial counts of flower food solution treatments over time (numbers in Iog10).

Treatment Day 2 Day 4 Day 6 Day 8

No microbial challenge

Water/sugar/citric ac.(WSC) 6.10 6.80 6.61 6.87

WSC/N-Ag 1.0 0.0 0.0 0.0 0.0

WSC/N-Ag 1.0/HQS 0.57 0.0 0.0 0.0

WSC/HQS 1.82 3.60 5.60 7.23

Microbial challenge

WSC/Ecc 6.49 7.39 7.70 7.52

WSC/N-Ag 1.0/Ecc 0.0 1.18 1.33 1.70

WSC/N-Ag 1.0/HQS/Ecc 0.67 0.0 0.0 0.0

WSC/HQS/Ecc 3.62 5.20 7.00 7.58

Under microbial challenge, the use of either silver or hydroxyquinoline alone increased the lifetime of these roses by about 55%. The use of N-Ag and HQS in combination completely eradicated microbial growth in vase water and increased the vase life of flowers by over 80% in comparison to untreated water. Further, the use of HQS at a concentration of 200 ppm in combination to Acticoat N-Ag enhanced the activity of either compound alone by about 50%. The results of these examples clearly indicate that the use of N-Ag produces a beneficial effect on fresh flower lifetime superior to that of HQS when no bacteria was inoculated to the mix. They showed the same results as biocides under bacterial challenge, differently from the two previous tests, where N-Ag was clearly superior under both set of treatments. Finally, a N-Ag and HQS combination (1.0 sq. inch Acticoat bandage, Hydroxyquinoline 200 ppm) showed strong synergistic effect on prolonging vase life and anti-microbial activity, indicating that the benefits produced by each compound alone are complementary and somewhat independent from one another.

EXAMPLE 6 Evaluation of the extent of bactericidal activity of two N-Ag powders (Nanotechnologies, N-Ag₃onm and N-Ag ₀₀nm) overtime in solution The objective of the study was to evaluate the longevity of continuous release and anti-microbial activity of silver ions when administered as nanocrystalline silver and to test the efficacy of another nanosilver compound than the one associated to the Acticoat bandage. Prior art has demonstrated that Acticoat N-Ag are continuously released and retain their antimicrobial activity for about 7 days. The killing activity of N-Ag powders carried in 30nm and 100nm particles was evaluated in vitro on Erwinia carotovora subsp. carotovora and Pseudomonas aeruginosa PA14, over period of three days. A two-fold dilution of N-Ag₃₀ and N-Agioo powder suspensions ranging from 1.0 - 1024.0 μg/mL were prepared in 96-well plates. Biofilms and planktonic cultures were grown in the MBEC Device for 8 h, then treated with N-Ag powders for 24h. After 24h, fresh biofilm and planktonic cultures were added to the same treatment solution, for another 24b, and so on. The treated cultures were recovered, serial diluted and plated to provide colony counts. The results demonstrate that N-Ag₃₀ and N-Ag₁₀o were still fairly active after 72h, under very challenging conditions (repeated inocula with fresh bacteria at each 24h). Results are summarized on Tables 13-16.

Table 13. Killing activity of N-Ag₃₀ on Erwinia carotovora planktonic and biofilm cuitures over a 72 h period. N-Ag₃₀ Planktonic Biofilm cone. μg/mL Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 0 7.00 7.60 7.90 4.10 4.20 4.30 1 7.00 7.48 8.17 4.30 3.95 4.87 2 7.60 7.00 8.11 4.13 3.67 3.77 4 1.00 6.78 8.04 3.63 3.10 3.12 8 0.00 0.00 7.70 0.00 0.00 1.16 16 0.00 0.00 5.00 0.67 0.33 0.67 32 0.00 0.00 0.00 0.00 0.00 0.00 64 0.00 0.00 0.00 0.77 0.50 0.93 128 0.00 0.00 0.00 0.00 0.00 0.00 256 0.00 o.oo 0.00 0.00 0.00 0.00 512 0.00 0.00 0.00 0.00 0.00 1.73 1024 0.00 0.00 0.00 0.00 0.00 0.67

Table 14. Killing activity of N-Ag₁₀₀ on Erwinia carotovora planktonic and biofilm cultures over a 72 h period. N-Ag₁₀o Planktonic Biofilm cone. μg/mL Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 0 7.00 7.00 7.78 4.84 4.40 4.69 1 0.00. 7.90 7.60 2.10 3.59 4.26 2 0.00 0.00 7.95 2.33 1.93 2.23 4 0.00 0.00 7.60 0.00 0.00 0.93 8 0.00 0.00 0.00 0.77 0.00 1.03 16 0.00 0.00 0.00 0.00 0.00 0.67 32 0.00 0.00 0.00 0.00 0.00 0.00 64 0.00 0.00 0.00 0.00 0.00 0.87 128 0.00 0.00 0.00 0.00 0.00 1.28 256 0.00 0.00 0.00 0.00 0.67 2.16 512 0.00 0.00 0.00 0.00 0.77 1.97 1024 0.00 0.00 0.00 0.00 1.59 1.93

Table 15. Killing activity of N-Ag₃₀ on Pseudomonas aeruginosa planktonic and biofilm cultures over a 72 h period. N-Ag₃₀ Planktonic Biofilm cone. μg/mL Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 0 7.00 7.48 8.00 6.00 6.23 6.00 1 7.17 7.70 8.53 5.81 5.73 5.28 2 0.00 7.30 7.70 5.37 5.13 5.30 4 0.00 7.60 4.00 5.56 5.00 5.46 8 0.00 0.00 0.00 5.43 5.06 5.38 16 0.00 0.00 0.00 5.54 5.07 4.85 32 0.00 0.00 0.00 5.10 5.28 5.32 64 0.00 0.00 0.00 4.89 5.10 5.59 128 0.00 0.00 0.00 4.71 4.67 5.12 256 0.00 0.00 0.00 4.15 4.37 5.11 512 0.00 0.00 0.00 4.24 4.16 4.00 1024 0.00 0.00 0.00 4.36 4.50 4.70 Table 16. Killing activity of N-Agι₀₀ on Pseudomonas aeruginosa planktonic and biofilm cultures over a 72 h period. N-Ag₁₀o Planktonic Biofilm cone. μg/mL Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 0 6.57 7.90 7.95 6.67 6.30 6.30 1 0.00 7.70 8.20 6.37 5.70 5.89 2 0.00 7.30 8.11 6.07 5.80 6.50 4 0.00 0.00 0.00 6.04 5.63 5.65 8 0.00 0.00 0.00 5.71 5.54 5.95 16 0.00 0.00 0.00 5.46 6.13 6.15 32 0.00 0.00 0.00 0.00 5.36 5.85 64 0.00 0.00 0.00 1.42 4.15 6.00 128 0.00 0.00 0.00 0.00 2.65 4.21 256 0.00 0.00 0.00 0.00 4.14 4.00 512 0.00 0.00 0.00 4.81 3.89 2.30 1024 0.00 0.00 0.00 4.69 3.09 4.98 These experiments demonstrate that N-Ag₃₀ and N-Agι₀o powders maintained good activity for at least three days, especially against bacteria in solution, including Erwinia and Pseudomonads species, two of the main groups of bacteria causing early senescence of cut flowers, under intense microbial challenge. The compounds were also effective against bJofjJms of Erwinia, which cause vascular disease in potato crops, along with post-harvest soft rot of many vegetables and ornamentals, seed potato spoilage, and premature death in cut flowers, indicating the potential of application of N-Ag as a prevention of treatment for these problems.

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Claims

WE CLAIM: 1. A method for treating plant material comprising contacting the plant material or a portion thereof with a composition that comprises at least one preservative agent, said preservative agent comprising nanocrystalline silver, thereby preserving the plant material. 2. The method of claim 1 wherein the preservative agent comprises a powder comprising nanocrystalline silver; a suspension comprising nanocrystalline silver; a solid matrix comprising nanocrystalline silver; a solution comprising nanocrystalline silver, or a container comprising nanocrystalline silver. 3. The method of claim 2 wherein the container comprises a bucket, box, holding container, storage container, shipping container, wrapper, paper wrapper, plastic wrapper, or foam. 4. The method of claim 1 wherein contacting a plant or portion thereof includes contacting a seed, root, stem, leaf, flower, and combinations thereof. 5. The method of claim 1 wherein contacting the plant material comprises contacting the plant material wftfi a one or more compositions selected from the group consisting of a harvesting composition, a hydrating composition, an impregnation composition, a pulsing composition, a storage composition, a bud-opening solution, and a biocide solution. 6. The method of claim 2 wherein contacting the plant material comprises contacting the plant material with a one or more compositions selected from the group consisting of a harvesting composition, a hydrating composition, an impregnation composition, a pulsing composition, a storage composition, a bud-opening solution, and a biocide solution. 7. The method of claim 1 further comprising contacting the plant with an ethylene inhibitor. 8. The method of claim 7 wherein the ethylene inhibitor comprises a hydroxyquinoline or ionic silver. 9. The method of claim 8 wherein the hydroxyquinoline is 8-hydroxyquinoline citrate or sulphate.

10. The method of claim 1 wherein preserving the plant material comprises extending the life of the plant material. 11. The method of claim 10 wherein extending the life of the plant material comprises extending the shelf life or vase life of a flower. 12. The method of claim 1 wherein preserving the plant material comprises treating the plant material against one or more biofilms. 13. The method of claim 12 wherein treating the plant material against one or more biofilms comprises eradicating or reducing the biofilm. 14. The method of claim 12 wherein treating the plant material against one or more biofilms comprises preventing the growth of a biofilm. 15. The method of claim 12 wherein treating the plant material against one or more biofilms comprises treating the plant material against one or more species selected from the group consisting of Erwinia carotovora, Pseudomonas syringae pv, phaseolicola, Pseudomonas aeruginosa, and sub-species and variants thereof. 16. The method of claim 1 wherein preserving the plant material comprises treating the plant material against one or more microorganisms. 17. The method of claim 12 wherein treating the plant material against one or more biofilms comprises eradicating or reducing the microorganisms. 8. The method of claim 12 wherein treating the plant material against one or more biofilms comprises preventing the growth of a microorganism. 19. A method for preserving plant material comprising contacting the plant material or a portion thereof with a composition that comprises at least one preservative agent, said preservative agent comprising nanocrystalline silver, thereby preserving the plant material. 20. The method of claim 19 wherein the preservative agent comprises a powder comprising nanocrystalline silver; a suspension comprising nanocrystalline silver; a solid matrix comprising nanocrystalline silver; a solution comprising nanocrystalline silver, or a container comprising nanocrystalline silver. 21. The method of claim 19 wherein contacting a plant or portion thereof includes contacting a seed, root, stem, leaf, flower, and combinations thereof.

22. A method of treating a plant material against a microorganism comprising contacting the plant material or a portion thereof with a composition that comprises at least one preservative agent, said preservative agent comprising nanocrystalline silver, thereby providing a benefit against said microorganism. 23. The method of claim 22 wherein the preservative agent comprises a powder comprising nanocrystalline silver; a suspension comprising nanocrystalline silver; a solid matrix comprising nanocrystalline silver; a solution comprising nanocrystalline silver, or a container comprising nanocrystalline silver. 24. The method of claim 23 wherein providing a benefit against said microorganism comprises eradicating, reducing, or preventing the microorganism. 25. A method of treating a plant material against a biofilm comprising contacting the plant material or a portion thereof with a composition that comprises at least one preservative agent, said preservative agent comprising nanocrystalline silver, thereby providing a benefit against said biofilm. 26. The method of claim 25 wherein the preservative agent comprises a powder comprising nanocrystalline silver; a suspension comprising nanocrystalline silver; a solid matrix comprising nanocrystalline silver; a solution comprising nanocrystalline silver, or a container comprising nanocrystalline silver. 27. The method of claim 25 wherein providing a benefit against said biofilm comprises eradicating, reducing, or preventing the biofilm. 28. The method of claim 25 further comprising first determining the presence of a biofilm. 29. The method of claim 28 further comprising determining and selecting one or more agents effective against said biofilm. 30. The method of claim 1 further comprising contacting the plant material during any stage of its processing. 31. The method of claim 22 further comprising contacting the plant material during any stage of its processing. 32. The method of claim 25 further comprising contacting the plant material during any stage of its processing.

33. The method of claim 30 wherein contacting the plant material comprises contacting the plant material at harvest in the field. 34. The method of claim 30 wherein contacting the plant material comprises contacting the plant material during pre-cooling. 35. The method of claim 30 wherein contacting the plant material comprises contacting the plant material during cold storage. 36. The method of claim 30 wherein contacting the plant material comprises contacting the plant material during transportation. 37. The method of claim 30 wherein contacting the plant material comprises contacting the plant material in the vase. 38. A method of claim 1 further comprising activating an automatic dispensing system to contact the plant material with the preservative composition. 39. The method of claim 1 further comprising contacting the plant material with a second preservative after treatment with the preservative composition. 40. The method of claim 39 wherein the second preservative comprises nanocrystalline silver or ionic silver. 41. The method of claim 5 wherein the harvesting composition comprises the anti-biofilm agent, water and an ethylene inhibitor. 42. The method of claim 5 wherein the hydrating composition comprises the anti-biofilm agent, water, an ethylene inhibitor, and a wetting agent. 43. The method of claim 5 wherein the impregnation composition comprises water and the anti-biofilm agent. 44. The method of claim 5 wherein the pulsing composition comprises the anti-biofilm agent and a nutrient. 45. The method of claim 5 wherein the bud opening composition comprises a nutrient, an anti-biofilm agent, and hormonal compounds. 46. A composition for treating and preserving plant material comprising a preservative agent comprising nanocrystalline silver and a carrier. 47. The composition of claim 46 wherein the composition further comprises one or more additional ingredients selected from the group consisting of an ethylene inhibitor, a nutrient, a germicide, a carbon source, a wetting agent, a water clarifier, and an acidifier. 48. The composition of claim 46 wherein the composition further comprises an ethylene inhibitor and a sugar source. 49. A composition for treating a biofilm comprising a preservative agent comprising nanocrystalline silver and a carrier. 50. The composition of claim 49 wherein the composition further comprises one or more additional ingredients selected from the group consisting of an ethylene inhibitor, a nutrient, a germicide, a carbon source, a wetting agent and an acidifier.

51. A method for preserving a plant material comprising contacting the plant material or a portion thereof with a composition comprising nanocrystalline silver; and treating a microbial infestation, wherein said microbial infestation comprises one or more microorganisms selected from the group consisting of Erwinia carotovora, Pseudomonas syringae pv, phaseolicola, Pseudomonas aeruginosa, and sub-species and variants thereof; and wherein treating said microbial infestation preserves or extends the shelf-life of the plant material. 52. The method of claim 51 wherein said treating a microbial infestation comprises eradicating, reducing, or preventing growth of the microorganism. 53. A method of treating a rose comprising contacting a rose plant or a portion thereof with a composition comprising nanocrystalline silver. 54. The method of claim 53 wherein treating a rose comprises treating a rose flower. 55. The method of claim 53 wherein treating a rose comprises treating a cut rose plant. 56. The method of claim 53 wherein the composition comprising nanocrystalline silver further comprises an ethylene inhibitor. 57. The method of claim 1 wherein the composition further comprises one or more additional ingredients selected from the group consisting of an ethylene inhibitor, a nutrient, a germicide, a carbon source, a wetting agent, a water clarifier, and an acidifier.