PLANTS

Technical Field

The present invention relates to a composition for controlling pathogenic microorganisms in plants, which includes nanosized silica-silver, and to a method of controlling pathogenic microorganisms in plants using the same.

Background Art

Generally, methods of controlling plant diseases by applying a predetermined material are largely classified into (1) direct disinfection methods to control diseases and (2) prevention methods using inductive resistance of plants (plant immunity) to control diseases. Most agricultural chemicals are concerned with methods of class (1) . Regarding methods of class (2) , methods of inducing an increase in disease resistance in plants using silicon (Si) (similar to the functions of immunity-enhancing material in animals) are provided. Silicon (Si) , which is the second most abundant material on the earth, is known to be absorbed into plants to increase disease resistance and stress resistance (Role of Root hairs and Lateral Roots in Silicon Uptake by Rice

J.F.Ma et al. Ichii Plant Physiology (2001) 127: 1773-

1780) . In particular, an aqueous silicate solution, used to treat plants, is reported to exhibit excellent preventive effects on pathogenic microorganisms chiefly responsible for causing powdery mildew or downy mildew in plants, and as well, is known to promote the physiological activity of plants, accelerating the growth of plants and inducing disease resistance and stress resistance in plants

(Suppressive effect of potassium silicate on powdery mildew of strawberry in hydroponics T. Kanto et al. J GenPlant Pathol (2004) 70: 207-211). However, since silica has no direct disinfection effects on pathogenic microorganisms in plants, it does not exhibit any effect on established diseases. Further, the effects of silica significantly vary with the physiological environment, and thus, they do not reach a predetermined level required for registration as an agricultural chemical (e.g., continuous exhibition of controlling effects on diseases of 80% or more) .

Silver (Ag) is known as a powerful disinfecting agent for killing unicellular microorganisms by inactivating enzymes having metabolic functions in the microorganisms by oligodynamic action (T. N. Kim, Q. L. Feng, et al. J. Mater. Sci. Mater. Med., 9, 129 (1998)). In addition to silver, although heavy metals, such as copper or zinc, may exert the same action, silver has the strongest antimicrobial effects. Also, silver is known to exhibit superb effects on algae. Research into silver as a

substitute for chlorine or other toxic microbicides has been continuously progressing. Moreover, various inorganic antimicrobial agents that use silver have been developed to date. Presently available silver-based inorganic antimicrobial agents are produced in the forms of silver- supported inorganic powders, silver colloids, metal silver powders, etc., of which silver-supported inorganic powders are the most used and thus are representative of a typical inorganic antimicrobial agent.

Silver in an ionic state is advantageous because it exhibits high antimicrobial activity. However, ionic silver is disadvantageous because it is unstable due to its high reactivity and thus may be easily oxidized or reduced into a metal depending on the surrounding atmosphere. Hence, silver causes discoloration by itself or allows other materials to cause undesirable coloration, and it does not continuously exert antimicrobial activity. Meanwhile, silver in the form of a metal or oxide is advantageous because it is stable in the environment, however it disadvantageous because it should be undesirably used in a relatively increased amount due to its low antimicrobial activity.

Silver, having the above advantages and disadvantages, is presently receiving attention in the form of nano-particles. Various methods of preparing the nano- particles include mechanical grinding, coprecipitation,

spraying, sol-gel manufacture, electrolysis, inverse microemulsion, etc. However, these methods are disadvantageous because the size of the particles formed is difficult to control, or high cost is required to prepare fine metal particles. For example, through the coprecipitation method, since the particles are prepared using an aqueous solution phase, the sizes, shapes and size distribution of the particles are impossible to control. Through the electrolysis and sol-gel methods, high preparation costs are required, and also, mass production is difficult. Although the inverse microemulsion method allows sizes, shapes and size distribution of the particles to be easily controlled, it may not be used in practice due to its complicated preparation processes. On the other hand, methods of preparing nanometer sized particles using exposure to radioactive rays are provided, which are advantageous because sizes, shapes and size distribution of the particles are easily controlled, and the particles may be prepared at room temperature. Also, preparation processes are simple, and therefore, mass production is possible at low costs.

Korean Patent No. 0425976 discloses a method of preparing a nanometer sized silver colloid using exposure to radioactive rays, and a nanometer sized silver colloid thus prepared. According to the above patent, a silver salt is dissolved in tertiary distilled water, added with sodium dodecylsulfate (SDS) , polyvinylalcohol (PVA) or

polyvinylpyrrolidone (PVP) as a colloid stabilizer, purged with nitrogen, and then exposed to radioactive rays, to prepare a silver colloid. However, the silver colloid thus prepared has a particle size of 100 run or larger, and must be used in a high concentration to exhibit antimicrobial actions on microorganisms, in particular, fungi.

In addition, Korean Patent Laid-open Publication No. 2003-0082065 (Application No. 10-2002-0020594) discloses a method of preparing a stable silver colloid, using a PVP used in Korean Patent No. 0425976, (1-vinylpyrrolidone) - acrylic acid copolymer, (1-vinylpyrrolidone) -vinylacetic acid copolymer, etc., as a polymer stabilizer.

In addition to the above methods, thorough attempts to provide a nano-silver having various functions such as antimicrobial action, purification and deodorization have been made. Still, less expensive methods of preparing stable nano-silver through a simpler process are required.

Under these circumferences, the present inventors have found that a silver salt, silicate and a water soluble polymer are mixed, and then exposed to radioactive rays, to prepare nanosized silica-silver particles comprising nano- silver that is combined with silica molecule and a water soluble polymer, which have a uniform size, are stable and exhibit excellent antimicrobial effects at a very low concentration, thereby completing the present invention.

Disclosure of the Invention

Accordingly, an object of the present invention is to provide a composition for controlling pathogenic microorganisms in plants, comprising nanosized silica- silver in which nano-silver is combined with silica molecule and water soluble polymer, as an effective component.

Another object of the present invention is to provide a method of controlling pathogenic microorganisms, using nanosized silica-silver in which nano-silver is combined with silica molecule and water soluble polymer.

Brief Description of the Drawings

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

FIG. Ia shows a schematic flowchart for the preparation of nanosized silica-silver, and FIG. Ib shows a transmission electron micrograph of the nanosized silica- silver; FIG. 2 shows the stability of colloidal nanosized silica-silver in an aqueous environment;

FIG. 3 shows the absorption spectrum (403 ran) of the nanosized silica-silver, water and silver ions;

FIG. 4 shows the absorbance (403 nm) of the nanosized

silica-silver varying with the concentration of sodium silicate (Na ₂ SiOs) ;

FIG. 5 shows the absorption spectrum (403 nm) of the nanosized silica-silver varying with the concentration of a water soluble polymer;

FIGS, βa and βb show the absorption spectrum (403 nm) of the nanosized silica-silver varying with the kind of water soluble polymer;

FIG. 7 shows the absorption spectrum (403 nm) of the nanosized silica-silver varying with the dose of radioactive rays;

FIG. 8a shows the antifungal effect of nanosized silica-silver on a pathogenic fungus in plants, Rhizoctonia solani, FIG. 8b shows the antifungal effect of nanosized silica-silver on Botrytis cinerea, and FIG. 8c shows the antifungal effect of nanosized silica-silver on Pythium ultimum and Magnaporthe grisea;

FIG. 9a shows the inhibitory effect of nanosized silica-silver on the growth of Escherichia coli, FIG. 9b shows the inhibitory effect of nanosized silica-silver on the growth of Bacillus subtilis 1021, FIG. 9c shows the inhibitory effect of nanosized silica-silver on the growth of

Pseudomonas syringae subsp. syringae 2440, FIG. 9d shows the inhibitory effect of nanosized silica-silver on the growth of Xanthomonas campestris pv. vesicatoria, FIG. 9e shows the inhibitory effect of nanosized silica-silver on the growth of

Azotobacter chrococuυm SL-206, and FIG. 9f shows the

inhibitory effect of nanosized silica-silver on the growth of Rhizobium tropici;

FIG. 10 shows the antifungal effect of nanosized silica-silver on young squash suffering from powdery mildew; FIG. 11 shows the antifungal effect of nanosized silica-silver on Phytophthora infestans;

FIG. 12 shows the chemical injuries caused by a high concentration of nanosized silica-silver on plants; and

FIG. 13 shows the inhibitory effect of nanosized silica-silver on spore germination of Botrytis cinerea and the inhibitory effect varying with a time period.

Best Mode for Carrying Out the Invention

According to an aspect, the present invention pertains to a composition for controlling pathogenic microorganisms in plants, comprising nanosized silica- silver in which nano-silver is combined with silica molecule and water soluble polymer, as an effective component.

In the present invention, the term "nanosized silica- silver" means a composite comprising nanosized silver particle and silica molecule that are combined with water soluble polymer. According to a specific aspect, nanosized silica-silver may be prepared by exposing a solution comprising silver salt, silicate and water soluble polymer to radioactive rays. One example of the composite is a

structure in which nanosized silver particles formed from silver ion and silica molecule formed from silicate are, individually or together, surrounded by water soluble polymer via exposure to radioactive rays. The nanosized silica-silver, in a colloidal state, may be present as nano-particles separated from each other or be formed into loose spherical clusters (FIG. Ib) . As such, the clusters may be simply separated when the temperature increases.

As confirmed in the absorption spectrum of FIG. 3, the nanosized silica-silver absorbs light of 403 nm which is the unique wavelength of nano-silver, and has a uniform nanoparticle size as shown in FIG. Ib. The particle size of the nanosized silica-silver preferably ranges from 0.5 to 30 nm, more preferably from 1 to 20 nm, and, most preferably, from 1 to 5 nm.

The nanosized silica-silver is obtained by preparing a solution comprising silver salt, silicate and water soluble polymer, and then exposing the solution to radioactive rays. This method further includes bubbling (or purging) the solution with an inert gas, before, after, or before and after exposing the solution to radioactive rays. The inert gas includes nitrogen, argon, etc., of which nitrogen gas may be preferably used. The bubbling is preferably performed for 10 to 30 min. When preparing the solution comprising silver salt, silicate and water soluble polymer, a radical scavenger is further included to scavenge radicals generated by exposure to radioactive rays. The radical scavenger

includes, for example, alcohol, glutathione, vitamin-E, flavonoid, ascrobic acid, etc. Usable alcohols are exemplified by methanol, ethanol, n-propanol, iso-propanol (IPA) , butanol, etc. Of these alcohols, iso-propanol may be preferably used. The alcohol may be used in an amount of 0.1 to 20%, and preferably, 3 to 10%, based on the total amount of the solution comprising silver salt, silicate and water soluble polymer.

Examples of the silver salt usable in the preparation of the nanosized silica-silver include silver nitrate (AgNO ₃ ) , silver perchlorate (AgClO ₄ ) , silver chlorate (AgClO ₃ ), silver chloride (AgCl), silver iodide (AgI), silver fluoride (AgF), silver acetate (CH ₃ COOAg), etc., of which a silver salt (e.g.: silver nitrate), easily soluble in water, may be preferably used. Examples of the water soluble polymer used in the preparation of the nanosized silica- silver include polyvinylpyrrolidone (PVP) , polyvinylalcohol (PVA) , polyacrylic acid and derivatives thereof, levan, flurane, gellane, water soluble cellulose, glucan, xanthane, water soluble starch, levan, corn starch, etc. Of these polymers, PVP may be preferably used. Examples of silicate used in the preparation of the nanosized silica-silver include sodium silicate, potassium silicate, calcium silicate, magnesium silicate, etc. Of these silicates, sodium silicate may be preferably used. Before disclosure in the present invention, the use of silicate in the preparation of nano-silver has not been found in any patent. The present

inventors first used silicate, not silica, to react with the silver salt, thereby providing nanosized silica-silver having high antimicrobial effects in which silica molecule and water soluble polymer are combined with nano-silver. Upon the preparation of nanosized silica-silver, the silver salt and silicate react in a weight ratio range of silver salt:silicate of 1:0.05 to 1.3. Preferably, the above two materials react at a weight ratio of 1:1. The particle size of nanosized silica-silver may be adjusted depending on the amount of silicate used. If silicate is used in a small amount, the particles become large. Meanwhile, if silicate is excessively used relative to the silver salt, the particles are not formed. Upon the preparation of nanosized silica-silver, the silver salt and the water soluble polymer react in a weight ratio range of silver salt:water soluble polymer of 1:0.5 to 2.5. Preferably, the above two materials react at a weight ratio of 1:1. To prepare the nanosized silica-silver, radioactive rays, such as beta rays, gamma rays, X-rays, ultraviolet light, electron rays, etc., may be used. Preferably, gamma rays may be used at a dose of 10 to 30 kGy.

In general, 1 to 5 nm sized particles may pass through a protoplasmic membrane, and silica is well absorbed into fungi. When the nanosized silica-silver is absorbed into fungal cells, silver nanoparticles function to increase disinfecting activity, and also, silica, which induces dynamic resistance to diseases to increase resistance, acts

to form a physical barrier to pathogenic fungi, and thus, recurrence of diseases may be prevented for a considerably long period after disinfection of pathogenic microorganisms.

Plants diseases are divided into 1) diseases caused by fungi, 2) diseases caused by prokaryotes, 3) diseases caused by parasites, 4) diseases caused by viruses and viroids, 5) diseases caused by nematodes, 6) diseases caused by protists, and 7) diseases caused by other non-biological causes. Among biological causes, plant diseases caused by fungi are the most common. These fungi have the following characteristics, that is, 1) large populations, 2) various forms of propagules (hyphae, conidiospores, ascospores, exotospores, aecidiospores, uredosproes, etc.), 3) various forms of bodies resistant to unfavorable environments (chlamydospores, oospores, sclerotia, etc.), and 4) easy variation due to a small genome size, and therefore, the fungi are difficult to control.

The composition for controlling pathogenic microorganisms in plants of the present invention may selectively control pathogenic fungi in plants at a very low concentration. In addition, when the composition is applied once, preventive effects may continue for 3 weeks or longer. The composition of the present invention can control both spores and hyphae, and has no chemical injury even if it is applied at a high concentration, and also, is harmless to the human body and to plants.

The nanosized silica-silver contained in the composition of the present invention has high preservability, and may be used in the state of being diluted in tap water or agricultural water. Compared to silver ions that are precipitated in the form of silver chloride along with chlorine ions in tap water, the nanosized silica-silver may be more easily handled and may reduce controlling costs.

Examples of pathogenic fungi in plants which may be treated and controlled using the nanosized silica-silver include Blumeria spp., Sphaerotheca spp., Phytophthora spp., Rhizoctonia spp., Fusariυm spp., Colletotrichum spp., Botrytis cinerea, Magnaporthe spp., Pythium spp., Puccinia spp., Erysiphe spp., Alternaria spp., Pseudoperonospora spp., Plasmodiophora spp., Sclerotinia spp., Fulvia spp., Peronospora spp., Ustilago spp., and Rtiizopus spp. In particular, the composition of the present invention exhibits excellent controlling effects on Blumeria spp., Sphaerotheca spp., Phytophthora spp., Rhizoctonia spp., Botrytis cinerea, and Pythium spp, among the above-mentioned pathogenic fungi. Examples of diseases caused by the above-mentioned pathogenic microorganisms include powdery mildew, late blight, Rhizoctonia disease, gray mold, blast, damping off, early blight, wilt, anthracnose, stem rot, Alternaria disease, Sclerotium disease, club root, seed rot, black rot, leaf spot, root rot, rusts, smuts, sooty mold, downy mildew, soft rot, brown patch, etc. These diseases may be effectively controlled using the nanosized silica-silver.

Powdery mildew is plant diseases that are the most common, visible to the naked eye, widely distributed, and easily recognizable, and damage almost all plants. Late blight causes various diseases to many plants ranging from seedlings of annual vegetables or ornamental plants to completely developed fruits and forest trees, and is typically represented by Phytophthora infestans. Rhizoctonia disease occurs over the whole world, and damages almost all vegetables and flowering plants, some cereals, lawn and perennial ornamental plants and trees, causing serious harm. In particular, brown patch causes lawn to brown and consequently wither. Thus, with the goal of controlling brown patch, many human and material resources are consumed. Gray mold commonly occurs in vegetables, ornamental plants, fruits, and even some upland crops, around the whole world, and is likely to be widely distributed. In addition, gray mold continuously grows and develops even during storage at 4 to 1O ⁰ C, thus decreasing the quality of fruits, and is also involved in putrefaction. Blast occurs throughout the whole world and is one of the most important diseases occurring in rice plants, and is one of the most important diseases occurring in rice growing areas where the use of a large amount of nitrogen fertilizer coincides with irrigation or heavy rainfall. In addition, damping-off of young seedlings is widely distributed over the whole world, and invades seeds, seedlings and roots of all plants. Although the grown plants seldom die even if suffering from damping-off,

damping-off progresses into laccolith or root rot of roots and land steins, and thus, remarkably retards plant growth and decreases the number of plants.

In particular, the composition of the present invention exhibits superb controlling effects on pathogenic fungi in plants even at a concentration of about 3 ppm, and also, excellent controlling effects on pathogenic fungi in plants even at a concentration of about 0.3 ppm. As shown in FIGS. 8b and 8c, growth and development of Botrytis cinerea and Pythium magnaporthe were inhibited at a concentration of 3 ppm. Further, as shown in FIGS. 8a and 10, growth and development of Rhizoctonia were inhibited at a concentration of 0.3 ppm, and powdery mildew was controlled.

In addition, the composition for controlling pathogenic microorganisms in plants of the present invention may control pathogenic bacteria in plants.

Examples of pathogenic bacteria in plants which may be controlled using nanosized silica-silver include Pseudomonas spp., Xanthomonas spp., Erwinia spp., Clavibacter spp., Ralstonia spp., Bacterium spp., Burkholderia spp., and Agrobacterium spp. In particular, the composition of the present invention exhibits excellent controlling effects on Pseudomonas spp., Xanthomonas spp., Ralstonia spp., and Burkholderia spp. , among the above-mentioned pathogenic bacteria in plants.

Examples of diseases caused by the above-mentioned pathogenic bacteria include leaf spot, bacterial blights,

wildfire, ring rot, canker, black rot, soft rot, galls, crown gall, scab, bacterial wilt, etc. These diseases may be effectively controlled using the nanosized silica-silver.

The composition of the present invention inhibits the growth and development of both Gram-positive bacteria and Gram-negative bacteria, in which the inhibitory effect on the growth and development of Gram-positive bacteria is higher than that on the growth and development of Gram-negative bacteria. It is preferable that the nanosized silica-silver contained in the controlling composition of the present invention have a particle size of 30 nm or less, preferably, 1 to 20 nm, and more preferably, 1 to 5 nm.

Although the composition of the present invention does not exhibit a controlling effect on bacteria at a low concentration, for example, 10 ppm, and particularly, 3 ppm or less, it exhibits a high controlling effect on fungi at the same concentration. Thus, when the composition of the present invention is applied at a concentration of 5 ppm or less, and preferably, 3 ppm, or in some cases, 0.3 ppm, it may selectively inhibit only pathogenic fungi in plants without action on bacteria beneficial to plants.

According to another aspect, the present invention pertains to a method of controlling pathogenic microorganisms in plants using the above composition.

The composition of the present invention may be mixed with an agriculturally acceptable carrier or diluent and thus

be formulated into various formulations including agricultural chemicals or fertilizers. In addition, the composition may be mixed with an additionally used fertilizer component or surfactant or known agents that control plant diseases. In the present invention, the term "diluent" means an agriculturally acceptable liquid or solid which is added to the nanosized silica-silver so that nanosized silica- silver is readily used or diluted at a desired active concentration. Examples of the diluent include talc, kaolin, zeolite, xylene, diatom, water, etc.

The formulation for use in a spray type, such as a water-dispersed concentrate or wet powder, may further include a wetting agent, a dispersant, a surfactant, etc. In addition to the diluent and the surfactant, a stabilizer, an inactivating agent, an adhesion improver, a colorant, an infiltrating agent, and a defoamer may be additionally included.

The composition of the present invention may be formulated into various forms. The wet powders prepared along with kaolin or diatoms are diluted with water before being used as a spraying liquid, and thus, may be sprayed onto leaves or applied to roots. Further, the composition may be mixed with an emulsifier to obtain a concentrate, which is then diluted with water before being applied to plants. Furthermore, the composition may be prepared into powders or granules through freeze drying, spray drying or rotary drying, packaged, transported, re-suspended by the end

user, and then applied to plants. As such, the composition may be sprayed onto leaves and/or stems and/or roots, or applied to soil, or may be granulated or encapsulated to be applied to soil. A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.

Example 1: Preparation Of Nanosized silica-silver Comprising Silica Molecule And Water Soluble Polymer

1 g of sodium silicate (Na ₂ Siθ ₃ ) , 1 g of silver nitrate (AgNO ₃ ) , 1 g of polyvinylpyrrolidone (PVP) , and 12 ml of isopropylalcohol (IPA) were added to distilled water and dissolved therein so that the total volume was 200 ml. The resultant solution was bubbled for 20 min using nitrogen gas, and then exposed to gamma rays of 25 kGy, thus preparing nanosized silica-silver.

FIG. Ia is a schematic flowchart showing the preparation of nanosized silica-silver comprising a silica molecule and a water soluble polymer, according to the present invention. The solution, formed after being exposed to gamma rays, was yellow due to nano-silver particles, which means that the silica molecule and the water soluble polymer combined with silver particle through the above reaction, yielding stable nanosized silica-silver particles.

To confirm whether the particles thus prepared were nano-silver particles, test groups as shown in Table 1, below, were prepared and allowed to stand at room temperature for 24 hr, after which color change was observed.

TABLE 1

* : Dissolved solution prepared in Example 1

The test groups A and B are solutions prepared by exposing the above dissolved solution to radioactive rays, and the test groups C and D are solutions in which Ag ⁺ ions were present without exposure to radioactive rays. The test groups SW and DW were control groups having no silver ions or silver particles.

Silver in the ionic state is easily oxidized, and precipitated in the form of AgCl while browning in the presence of Cl ^" ions. Thus, the state of silver may be confirmed using tap water having Cl ^" ions. In the case where silver is present in the state of Ag ⁺ ions, it precipitates. Meanwhile, when silver is present in stable nano-silver particles, it shows yellow. The results are

given in Table 2, below.

TABLE 2

As is apparent from Table 2, the test groups SW, D and DW were colorless without color change even after allowing to stand for 24 hr, which means that silver ions, chlorine ions, or silver ions and chlorine ions were all absent. However, the colorless test group C turned to reddish brown, which means that silver ions were formed into AgCl along with chlorine ions in tap water. In addition, the test groups A and B showed yellow without color change, which means that stable nano-silver particles combined with a silica molecule and a water soluble polymer were formed via exposure to radioactive rays and AgCl precipitates were not formed even in the presence of chlorine ions. The color change is shown in FIG. 2.

The absorption spectrum of the nanosized silica- silver of the present invention is shown in FIG 3. FIG. 3 shows the absorption spectrum of the solutions of the test groups DW, B and D in Table 2, in which only the test group B absorbed light of 403 nm, which is the unique wavelength

of nano-silver, and the test groups DW and D did not absorb light at the same wavelength.

From the result of the absorption spectrum, measured after the solution was allowed to stand, it was confirmed that stable nanosized silica-silver particles comprising silica molecule and water soluble polymer were formed by exposing the solution comprising sodium silicate, silver nitrate and PVP to radioactive rays.

FIG. Ib shows a photograph of the prepared nanosized silica-silver, observed using a transmission electron microscope (TEM) . As shown in FIG. Ib, the nanosized silica-silver particles have uniform particle size distribution having a particle size of 1 to 5 nm smaller than 20 nm. The nanosized silica-silver particles may be independently separated or be formed into loose spherical clusters due to intermolecular attraction. As such, the clusters may be easily separated by heat.

Example 2: Preparation Of Nanosized silica-silver Comprising Silica Molecule And Water Soluble Polymer

Nanosized silica-silver was prepared in the same manner as in Example 1, with the exception of varying the concentration of sodium silicate (Na ₂ SiC> ₃ ) from 0.5 to 2 g.

The test groups having different concentrations are shown in Table 3, below.

TABLE 3

The absorbance and color of nanosized silica-silver varying with the concentration of sodium silicate as shown in Table 3 are shown in FIG. 4.

As shown in FIG. 4, when sodium silicate and silver nitrate are mixed at a ratio of 1:1, the highest absorbance is obtained. When sodium silicate and silver nitrate are mixed at a ratio of at least 1.5:1, the absorbance is somewhat decreased. Meanwhile, when sodium silicate and silver nitrate are mixed at a ratio of 0.5 or less:l, the size of the silver particles is increased as evidenced by an orange gold color.

As is apparent from the above result, since the amount of sodium silicate functions as an important factor to prepare nanosized silica-silver, it may be adjusted to regulate the particle size of the nanosized silica-silver.

Example 3: Preparation Of Nanosized silica-silver Comprising Silica Molecule And Water Soluble Polymer

Nanosized silica-silver was prepared in the same manner as in Example 1, with the exception of varying the concentration of polyvinylpyrrolidone (PVP) from 0.5 to 2

The absorbance and color of nanosized silica-silver varying with the concentration of PVP are shown in Table 4, below, and FIG. 5.

TABLE 4

As shown in Table 4 and FIG. 5, when sodium silicate and silver nitrate are mixed at the same ratio, polyvinylpyrrolidone (PVP) may be used at a concentration 0.5 to 2 times that of sodium silicate (or silver nitrate).

Example 4: Preparation Of Nanosized silica-silver Comprising Silica Molecule And Water Soluble Polymer

Nanosized silica-silver was prepared in the same manner as in Example 1, with the exception of using high levan or corn starch, instead of polyvinylpyrrolidone (PVP) . The absorbance and absorption spectrum of the prepared nanosized silica-silver are shown in Table 5, below, and FIG. 6.

TABLE 5

As shown in Table 5 and FIG. 6, the nanosized silica- silver may be prepared using polysaccharides such as levan or corn starch, although it has lower absorbance than nanosized silica-silver prepared using polyvinylpyrrolidone (PVP) .

Example 5: Preparation Of Nanosized silica-silver Comprising Silica Molecule And Water Soluble Polymer Nanosized silica-silver was prepared in the same manner as in Example 1, with the exception of varying the dose of radioactive rays.

The absorbance and absorption spectrum of the prepared nanosized silica-silver are shown in Table β, below, and FIG. 7.

TABLE 6

As seen in Table 6 and FIG. 7, absorption occurred even at 10 kGy, and increased as the dose of radioactive rays was increased. Thus, the nanosized silica-silver may be prepared using radioactive rays of 10 kGy or more.

Experimental Example: Antimicrobial Effects Of Nanosized silica-silver Comprising Silica Molecule And Water Soluble Polymer To confirm antimicrobial effects of nanosized silica- silver comprising silica molecule and water soluble polymer, antimicrobial effects on various pathogenic microorganisms were measured.

Experimental Example 1: Antifungal Effect On Rhlzoctonia

A microorganism culture medium (PDA medium, Difco Co. Ltd. ) was autoclaved, and then 25 ml were aliquotted into each of a plurality of petri dishes. Before the aliquotted medium was cured (at about 40 ⁰ C) , it was mixed with silica from the test group A, mixed with the nanosized silica- silver prepared in Example 1 from the test group B, mixed with 20 nm sized silver particles from the test group C, and mixed with 100 nm sized silver particles from the test group D, and then cooled, to prepare each medium. Into the prepared medium, a solid medium having sufficiently cultured Rhizoctonia solan! as one of the pathogenic fungi in plants, which was removed on a circular piece having a diameter of 5 mm, was inoculated. Through culture at room temperature for 2 days, whether the growth of the microorganism had been inhibited was confirmed. The concentration of the mixed material of each test group was

set to 6 ppm and 0. 3 ppm.

As shown in FIG. 8a, the test group A including only silica had the same result as a control group, regardless of the concentration. The test groups C and D including 20 run sized silver and 100 nm sized silver, respectively, had the same results as a control group, at a concentration of

0.3 ppm. However, the test group B including the nanosized silica-silver of the present invention exhibited an excellent inhibitory effect on the growth of Rhizoctonia solani, even at a low concentration of 0.3 ppm.

Experimental Example 2: Antifungal Effect On Botrytis

A microorganism culture medium (PDA medium, Difco Co.

Ltd. ) was autoclaved, and then 25 ml were aliquotted into each of a plurality of petri dishes. Before the aliquotted medium was cured (at about 4O ⁰ C) , it was mixed with silica from the test group A, mixed with the nanosized silica- silver prepared in Example 1 from the test group C, mixed with 20 nm sized silver particles from the test group B, and mixed with 100 nm sized silver particles from the test group D, and then cooled, to prepare each medium. Into the prepared medium, a solid medium having sufficiently cultured Botrytis clnerea as one of the pathogenic fungi in plants, which was removed on a circular piece having a diameter of 5 mm, was inoculated. Through culture at room temperature for 2 days, whether the growth of the microorganism had been inhibited was confirmed. The

concentration of the mixed material of each test group was set to β ppm, 3 ppm, and 0.3 ppm.

As shown in FIG. 8b, the test group A including only silica had the same result as a control group, regardless of the concentration. The test groups B and D, including 20 nm sized silver and 100 nm sized silver, respectively, had the same results as a control group at a concentration of 0.3 ppm. However, the test group C including the nanosized silica-silver of the present invention had a higher inhibitory effect on the growth of Botrytis cinerea than those of test groups including 20 nm sized silver and 100 nm sized silver, even at a low concentration of 3 ppm.

Further, as seen in FIG. 13, the nanosized silica- silver inhibited spore germination of Botrytis cinerea at a low concentration of 3 ppm.

Experimental Example 3: Antifungal Effects On Pythium And Magnaporthe

A microorganism culture medium (PDA medium, Difco Co. Ltd. ) was autoclaved, and then 25 ml were aliquotted into each of a plurality of petri dishes. Before the aliquotted medium was cured (at about 40°C) , it was mixed with the nanosized silica-silver prepared in Example 1 at a concentration of 0, 0.3, 1.6, and 3 ppm, and then cooled, to prepare each medium. Into the prepared medium, a solid medium having sufficiently cultured Pythium ultimum or Magnaporthe grisea, which was removed on a circular piece

having a diameter of 5 mm, was inoculated. Through culture at room temperature for 2 days, whether the growth of the microorganisms had been inhibited was confirmed.

As shown in FIG. 8c, the nanosized silica-silver of the present invention exhibited a remarkably high inhibitory effect on the growth of Pythlum ultimum and Magnaporthe grisea at 3 ppm.

Experimental Example 4: Antifungal Effect On Powdery Mildew

To assay controlling effects of the nanosized silica- silver prepared in Example 1 on pathogenic fungi in plants, the present experiment was carried out in a plastic film greenhouse of young squash plants infected with powdery mildew. 0.3 ppm nanosized silica-silver was uniformly applied onto young squash plants infected with powdery mildew. After the nanosized silica-silver was applied, the state of powdery mildew was observed for 3 weeks.

FIG. 10 is photographs showing the controlling effects on powdery mildew, 0, 3 and 7 days after the nanosized silica-silver was applied. Although powdery mildew was widespread on the leaves of young squash at day 0, controlling effects close to 100% were exhibited after the nanosized silica-silver was applied. After about 3 weeks, powdery mildew was not observed.

Experimental Example 5: Antifungal Effect On

Phytophthora infestans

The controlling effect of the nanosized silica-silver prepared in Example 1 on pathogenic fungi in plants was assayed using potatoes (cv. Desiree) infected with Phytophthora spp.

The upper surface of the potato leaf was coated with lens-paper, and then inoculated with zoospores of Phytophthora infestans at a concentration of 3 x 10 ⁴ zoospores/ml. Thereafter, humidity of 95% or more was maintained. 3 days after Phytophthora infestans was inoculated, the nanosized silica-silver was diluted 1000 times and then sprayed onto the potato leaf.

FIG. 11 is photographs showing the results of spray treatment 3 days after inoculation and the controlling effects 6 and 9 days after the spray treatment. From the photographs of FIG. 11, the nanosized silica-silver was confirmed to exhibit excellent controlling effect on Phytophthora infestans.

Experimental Example 6: Antibacterial Effects of Nanosized silica-silver

To assay the inhibitory effects on the growth of bacteria varying with the concentration of nano-silver which was combined with silica molecule and water soluble polymer, Escherichia coli _r Bacillus subtilis 1021, Pseudomonas syringae subsp. syringae 2440, Xanthomonas campestris pv. vesicatoria, Azotobacter chrococuum SL206,

and Rhizobium tropici were used. After loading 100 ml of LB medium into a 500 ml Erlenmeyer flask, Escherichia coli was cultured at 37 ⁰ C, and the other bacteria were cultured at 30°C, for 15 to 16 hrs under aerobic condition on rotary shaker at 190 rpm. After culture, 20 μl of culture fluid of each strain were inoculated into an LB agar plate containing nanosized silica-silver comprising a silica molecule and a water soluble polymer at concentrations of 0, 1, 10, 100, and 1000 ppm. Subsequently, Escherichia coli was cultured at 37°C, and the other bacteria were cultured at 3O ⁰ C, for 6 to 7 days.

The inhibitory effect on the growth of Escherichia coli is shown in FIG. 9a, the inhibitory effect on the growth of Bacillus subtilis 1021 in FIG. 9b, the inhibitory effect on the growth of Pseudomonas syringae subsp. syringae 2440 in FIG. 9c, the inhibitory effect on the growth of Xanthomonas campestris pv. vesicatoria in FIG. 9d, the inhibitory effect on the growth of Azotobacter chrococuum SL206 in FIG. 9e, and the inhibitory effect on the growth of Rhizobium tropici in FIG. 9f.

The growth of Bacillus subtilis 1021, a Gram-positive bacteria, was decreased at 10 ppm, compared to a control group (LB agar plate) . On the other hand, Escherichia coli

(Probe, PR2) , Pseudomonas syringae subsp. syringae 2440, Xanthomonas campestris pv. vesicatoria, Azotobacter chrococuum SL206, and Rhizobium tropici, Gram-negative bacteria, grew similarly on the control group (LB agar

plate) , the medium containing 1 ppm nanosized silica- silver, and the medium containing 10 ppm nanosized silica- silver. In particular, the growth of all strains was completely inhibited on the medium containing 100 ppm nanosized silica-silver.

From the above results, it can be found that the growth of bacteria is inhibited using the nanosized silica- silver at a high concentration (100 ppm) , whereas the growth of fungi is inhibited using 3 ppm nanosized silica- silver. Hence, nanosized silica-silver having a low concentration is used to control pathogenic fungi in plants, while nanosized silica-silver having a higher concentration than the nanosized silica-silver used for control of the fungi is used to control bacteria.

Experimental Example 7: Chemical Injuries From Nanosized silica-silver At High Concentration On Plants

To assay the chemical injuries due to nanosized silica-silver having a high concentration on plants, an undiluted solution and 10, 100, and 1000 times diluted solutions of nanosized silica-silver were each prepared. These undiluted solution and diluted solutions were applied on the surfaces of leaves including new leaves of pepper (Ground Cherry, Hungnong) , lettuce (Red Chima, Kwonnong) , cucumber (Joenbaecdadaki, Hungnong) , tomato (Seokwang, Hungnong) , paprika (Special Yellow, Netherlands) , Korean cabbage (Mae Leok, Nongwoo) , chicory (Red, Jeil) , kale

(Green Kale, Nongwoo) , mini tomato (Kyoko, Dakei) and pansy (Magestic Giant Yellow, Sakada) . 3 days after the nanosized silica-silver was applied, chemical injuries on plants were observed. FIG. 12 shows the chemical injuries of nanosized silica-silver applied at a high concentration on the entire surfaces of leaves including new pepper leaves. Compared to a control group, typical chemical injury phenomena, such as wrinkle of new leaves, did now show in the treatment groups using the diluted solutions and the undiluted solution. These results were the same as when applied to other plants.

Industrial Applicability

As described above, the present invention provides a composition for controlling pathogenic microorganisms in plants. Nanosized silica-silver of the present invention exhibits a wide range of antimicrobial activity, and can control both spores and hyphae. In addition, the nanosized silica-silver manifests efficient controlling effects at low concentrations, and may maintain the controlling effects for a long period upon a single application.

Further, even if the nanosized silica-silver is used at a high concentration, it does not cause chemical injuries and is nontoxic to the human body and to plants. Moreover, the nanosized silica-silver can selectively control the

microorganisms depending on its concentration.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.