Application of composition for preventing and treating tobacco angular leaf spot disease in enhancing resistance of tobacco to tobacco angular leaf spot disease

Abstract

The invention provides application of a composition for preventing and treating tobacco angular leaf spot in enhancing resistance of tobacco to tobacco angular leaf spot. The composition comprises nano titanium dioxide, nano copper hydroxide and nano silver. The invention creatively finds that the nano titanium dioxide, the nano copper hydroxide and the nano silver have a synergistic effect in the aspect of enhancing the resistance of tobaccos to the tobacco angular leaf spot germs, and the synergistic effect of the nano titanium dioxide, the nano copper hydroxide and the nano silver has a remarkable inhibition effect on the tobacco angular leaf spot germs and has an excellent prevention and treatment effect on the tobacco angular leaf spot germs; the composition has the advantages of safe components, no pollution to soil environment, excellent prevention and treatment effect on tobacco angular leaf spot, reduction of the incidence rate of tobacco field diseases, and improvement of the quality and yield of tobaccos.

Description

Technical Field

[0001] The invention belongs to the technical field of tobacco disease prevention and control, and particularly relates to an application of a composition for preventing and treating tobacco angular leaf spot in enhancing tobacco resistance to tobacco angular leaf spot pathogens. Background Art

[0002] Tobacco angular leaf spot, commonly known as black fire, is caused by infection with Pseudomonas syringae pv. tabaci, a Gram-negative pathogen. It is a common bacterial disease in tobacco fields, severely impacting tobacco yield. Angular leaf spot can occur at any stage of tobacco production, but is more common in the middle and late stages. Leaves, stems, flowers, and capsules of tobacco plants are susceptible, primarily on the leaves. Angular leaf spot first appears on the base of leaves. Lesions begin as small, oily spots, then expand into polygonal or irregular spots. These spots are bounded by leaf veins, with distinct dark brown to blackish brown margins. Sometimes the center of the spot is uneven in color, often exhibiting gray-brown cloud-like patterns. Lesions are large and can coalesce to form a large angular spot, surrounded by a less distinct halo. Leaf veins can also turn brown due to infection, expanding along the veins to form streaks.

[0003] First reported in North Carolina, USA, in 1917, tobacco angular leaf spot has now been reported in major tobacco-producing regions worldwide, becoming a serious threat to tobacco production. In recent years, factors such as the increasing age of tobacco varieties and poor agricultural practices have created conditions for the disease to become prevalent in fields, leading to a growing severity of the disease and significant economic losses. However, effective technical measures to control tobacco angular leaf spot remain lacking, resulting in severe disease outbreaks in tobacco fields and a significant reduction in tobacco quality and yield. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a composition for preventing and treating tobacco angular spot disease and its application in enhancing tobacco's resistance to tobacco angular spot disease bacteria. The present invention creatively discovered that nano-titanium dioxide, nano-copper hydroxide and nano-silver have excellent prevention and treatment effects on tobacco angular spot disease, significantly reducing the incidence of diseases in tobacco fields and improving tobacco quality and yield.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides an application of a composition for preventing and treating tobacco angular leaf spot in enhancing tobacco resistance to tobacco angular leaf spot pathogens, wherein the composition comprises nano-titanium dioxide, nano-copper hydroxide and nano-silver.

[0007] The present invention creatively discovers that nano-titanium dioxide, nano-copper hydroxide and nano-silver have a synergistic effect in enhancing tobacco's resistance to tobacco angular leaf spot pathogen. The three work together to have a significant inhibitory effect on tobacco angular leaf spot pathogen and have excellent preventive and therapeutic effects on tobacco angular leaf spot. The ingredients are safe and pollution-free to the soil environment. They have excellent preventive and therapeutic effects on tobacco angular leaf spot pathogen, reduce the incidence of diseases in tobacco fields, and improve tobacco quality and yield.

[0008] Preferably, the composition comprises 30-55 parts of nano titanium dioxide, 10-34 parts of nano copper hydroxide and 5-20 parts of nano silver in parts by weight.

[0009] The weight proportions of the nano-titanium dioxide may be 30 parts, 32 parts, 35 parts, 38 parts, 40 parts, 42 parts, 45 parts, 48 parts, 50 parts, 52 parts or 55 parts.

[0010] The weight proportions of the nano copper hydroxide may be 10 parts, 12 parts, 15 parts, 18 parts, 20 parts, 22 parts, 25 parts, 28 parts, 30 parts, 32 parts or 34 parts.

[0011] The weight proportions of the nanosilver may be 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, 19 parts or 20 parts, etc.

[0012] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0013] The nano-titanium dioxide, nano-copper hydroxide and nano-silver in the present invention have a good inhibitory effect on tobacco angular leaf spot pathogens under the above-mentioned specific content ratio, and have a good prevention and control effect on tobacco angular leaf spot disease.

[0014] Preferably, the composition comprises 35-45 parts of nano titanium dioxide, 15-25 parts of nano copper hydroxide and 10-15 parts of nano silver in parts by weight.

[0015] The weight proportions of the nano titanium dioxide may be 35 parts, 36 parts, 37 parts, 38 parts, 39 parts, 40 parts, 41 parts, 42 parts, 43 parts, 44 parts or 45 parts.

[0016] The weight proportions of the nano copper hydroxide may be 15 parts, 16 parts, 17 parts, 18 parts, 19 parts, 20 parts, 21 parts, 22 parts, 23 parts, 24 parts or 25 parts, etc.

[0017] The weight proportions of the nanosilver may be 10 parts, 10.5 parts, 11 parts, 11.5 parts, 12 parts, 12.5 parts, 13 parts, 13.5 parts, 14 parts, 14.5 parts or 15 parts, etc.

[0018] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0019] Furthermore, the nano-titanium dioxide, nano-copper hydroxide and nano-silver in the present invention have the best inhibitory effect on tobacco angular leaf spot pathogens under the above-mentioned specific content ratio, and have the best prevention and treatment effect on tobacco angular leaf spot disease.

[0020] Preferably, the particle size of the nano-titanium dioxide, nano-copper hydroxide and nano-silver is independently 1-100 nm, for example, 1 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm or 100 nm.

[0021] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0022] In a second aspect, the present invention provides a fungicide for preventing and treating tobacco angular spot disease, the fungicide comprising a composition for preventing and treating tobacco angular spot disease and an agriculturally acceptable functional adjuvant; the composition comprises nano-titanium dioxide, nano-copper hydroxide and nano-silver.

[0023] The fungicide provided by the present invention has safe ingredients, is non-polluting to the soil environment, and exhibits excellent control effects against tobacco angular leaf spot, reducing the incidence of tobacco field diseases and improving tobacco quality and yield. The present invention innovatively discovered that nano-titanium dioxide, nano-copper hydroxide, and nano-silver exhibit a synergistic effect in enhancing tobacco's resistance to the pathogen Angular Leaf Spot. The three agents work together to significantly inhibit the pathogen, providing excellent preventive and therapeutic effects against the disease.

[0024] Preferably, the composition comprises 30-55 parts of nano titanium dioxide, 10-34 parts of nano copper hydroxide and 5-20 parts of nano silver in parts by weight.

[0025] The weight proportions of the nano-titanium dioxide may be 30 parts, 32 parts, 35 parts, 38 parts, 40 parts, 42 parts, 45 parts, 48 parts, 50 parts, 52 parts or 55 parts.

[0026] The weight proportions of the nano copper hydroxide may be 10 parts, 12 parts, 15 parts, 18 parts, 20 parts, 22 parts, 25 parts, 28 parts, 30 parts, 32 parts or 34 parts.

[0027] The weight proportions of the nanosilver may be 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, 19 parts or 20 parts, etc.

[0028] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0029] The nano-titanium dioxide, nano-copper hydroxide and nano-silver in the present invention have a good inhibitory effect on tobacco angular leaf spot pathogens under the above-mentioned specific content ratio, and have a good prevention and control effect on tobacco angular leaf spot disease.

[0030] Preferably, the composition comprises 35-45 parts of nano titanium dioxide, 15-25 parts of nano copper hydroxide and 10-15 parts of nano silver in parts by weight.

[0031] The weight proportions of the nano titanium dioxide may be 35 parts, 36 parts, 37 parts, 38 parts, 39 parts, 40 parts, 41 parts, 42 parts, 43 parts, 44 parts or 45 parts.

[0032] The weight proportions of the nano copper hydroxide may be 15 parts, 16 parts, 17 parts, 18 parts, 19 parts, 20 parts, 21 parts, 22 parts, 23 parts, 24 parts or 25 parts, etc.

[0033] The weight proportions of the nanosilver may be 10 parts, 10.5 parts, 11 parts, 11.5 parts, 12 parts, 12.5 parts, 13 parts, 13.5 parts, 14 parts, 14.5 parts or 15 parts, etc.

[0034] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0035] Furthermore, the nano-titanium dioxide, nano-copper hydroxide and nano-silver in the present invention have the best inhibitory effect on tobacco angular leaf spot pathogens under the above-mentioned specific content ratio, and have the best prevention and treatment effect on tobacco angular leaf spot disease.

[0036] Preferably, the mass percentage of the composition in the fungicide is 10-95%, for example, it can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

[0037] Preferably, the mass percentage of the composition in the fungicide is 22-86%, for example, it can be 22%, 25%, 35%, 45%, 55%, 65%, 75%, 85% or 86%.

[0038] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0039] Preferably, the agriculturally acceptable functional adjuvant includes any one or a combination of at least two of a solvent, an emulsifier, a dispersant, a wetting agent, an adhesive, a stabilizer, a defoaming agent, a diluent, a thickener or an antifreeze agent.

[0040] Preferably, the dosage form of the fungicide includes any one of powder, suspension, microemulsion, ointment, plaster or spread.

[0041] Preferably, the fungicide is applied by any one of foliar spraying, soil mixing, root irrigation, seed soaking, furrow application or hole application, or a combination of at least two of them.

[0042] In a third aspect, the present invention provides a method for preventing and controlling tobacco angular spot disease, comprising applying the fungicide of the second aspect to tobacco and / or its cultivation medium before or after tobacco is infected.

[0043] Preferably, nano-titanium dioxide, nano-copper hydroxide and nano-silver are applied simultaneously or sequentially.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The present invention creatively discovers that nano-titanium dioxide, nano-copper hydroxide and nano-silver have a synergistic effect in enhancing tobacco's resistance to tobacco angular leaf spot pathogen. The three work together to have a significant inhibitory effect on tobacco angular leaf spot pathogen and have excellent preventive and therapeutic effects on tobacco angular leaf spot. The ingredients are safe and pollution-free to the soil environment. They have excellent preventive and therapeutic effects on tobacco angular leaf spot pathogen, reduce the incidence of diseases in tobacco fields, and improve tobacco quality and yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Pictures of tobacco leaf spots on tobacco K326 after applying the compositions prepared in Example 1 or Comparative Examples 1-3 and then inoculating with Nicotiana tabacum. DETAILED DESCRIPTION

[0047] In order to further illustrate the technical means and effects adopted by the present invention, the technical solutions of the present invention are further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0048] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0049] The sources of materials used in the following specific embodiments are as follows:

[0050] Raw material name Purchase manufacturer model Nano-titanium dioxide Solarbio NM000800 Nano copper hydroxide Shanghai Chengxin Industrial Co., Ltd. 20427-59-2 Nanosilver Solarbio NM000620 Copper hydroxide Acmec C36540-500g Nano copper oxide Acmec C49342-25g Nano zinc oxide Acmec Z86341-100g Nano-alumina Acmec A26796-100g Nano-cobalt oxide Aladdin C111615-5g Nano-cerium oxide Aladdin C103984-500g

[0051] Example 1

[0052] This embodiment provides a composition for preventing and treating tobacco angular spot, which comprises, by weight, 40 parts of nano-titanium dioxide, 20 parts of nano-copper hydroxide, and 12 parts of nano-silver. The preparation method is to mix the above components evenly.

[0053] Example 2

[0054] This embodiment provides a composition for preventing and treating tobacco angular spot, which comprises, by weight, 35 parts of nano-titanium dioxide, 25 parts of nano-copper hydroxide, and 15 parts of nano-silver. The preparation method is to mix the above components evenly.

[0055] Example 3

[0056] This embodiment provides a composition for preventing and treating tobacco angular spot, which comprises, by weight, 45 parts of nano-titanium dioxide, 15 parts of nano-copper hydroxide, and 10 parts of nano-silver. The preparation method is to mix the above components evenly.

[0057] Example 4

[0058] This embodiment provides a composition for preventing and treating tobacco angular spot, which comprises, by weight, 30 parts of nano-titanium dioxide, 34 parts of nano-copper hydroxide, and 20 parts of nano-silver. The preparation method is to mix the above components evenly.

[0059] Example 5

[0060] This embodiment provides a composition for preventing and treating tobacco angular spot, which comprises, by weight, 55 parts of nano-titanium dioxide, 10 parts of nano-copper hydroxide, and 5 parts of nano-silver. The preparation method is to mix the above components evenly.

[0061] Example 6

[0062] This embodiment provides a composition for preventing and treating tobacco angular spot, which comprises, by weight, 7 parts of nano-titanium dioxide, 40 parts of nano-copper hydroxide, and 25 parts of nano-silver. The preparation method is to mix the above components evenly.

[0063] Example 7

[0064] This embodiment provides a composition for preventing and treating tobacco angular spot, which comprises, by weight, 66 parts of nano-titanium dioxide, 5 parts of nano-copper hydroxide, and 1 part of nano-silver. The preparation method is to mix the above components evenly.

[0065] Comparative Example 1

[0066] This comparative example provides a composition for preventing and treating tobacco angular spot disease, which differs from Example 1 only in that nano-titanium dioxide is not added, and its reduced amount is proportionally distributed to nano-copper hydroxide and nano-silver.

[0067] Comparative Example 2

[0068] This comparative example provides a composition for preventing and treating tobacco angular spot disease, which differs from Example 1 only in that nano-copper hydroxide is not added, and its reduced amount is proportionally distributed to nano-titanium dioxide and nano-silver.

[0069] Comparative Example 3

[0070] This comparative example provides a composition for preventing and treating tobacco angular spot, which differs from Example 1 only in that nano-silver is not added, and its reduced amount is proportionally distributed to nano-titanium dioxide and nano-copper hydroxide.

[0071] Comparative Example 4

[0072] This comparative example provides a composition for preventing and treating tobacco angular spot disease, which differs from Example 1 only in that nano copper hydroxide is replaced with an equal portion of copper hydroxide.

[0073] Comparative Example 5

[0074] This comparative example provides a composition for preventing and treating tobacco angular spot disease, which differs from Example 1 only in that nano copper hydroxide is replaced by an equal portion of nano copper oxide.

[0075] Comparative Example 6

[0076] This comparative example provides a composition for preventing and treating tobacco angular spot disease, which differs from Example 1 only in that nano copper hydroxide is replaced by an equal portion of nano zinc oxide.

[0077] Comparative Example 7

[0078] This comparative example provides a composition for preventing and treating tobacco angular spot disease, which differs from Example 1 only in that nano copper hydroxide is replaced by an equal portion of nano aluminum oxide.

[0079] Comparative Example 8

[0080] This comparative example provides a composition for preventing and treating tobacco angular spot disease, which differs from Example 1 only in that nano titanium dioxide is replaced by an equal portion of nano cobalt oxide.

[0081] Comparative Example 9

[0082] This comparative example provides a composition for preventing and treating tobacco angular spot disease, which differs from Example 1 only in that nano-titanium dioxide is replaced by an equal portion of nano-cerium oxide.

[0083] Comparative Example 10

[0084] This comparative example provides a commercially available tobacco fungicide.

[0085] Test Example 1

[0086] Pseudomonas syringae pv. angulata (kindly provided by Zhengzhou Tobacco Science Research Institute) was cultured in LB liquid medium with shaking at 28°C overnight. The bacterial solution was centrifuged at 5000 rpm for 10 min, and the bacterial pellet was resuspended in PBS solution for washing. The pellet was centrifuged again and resuspended in PBS solution to prepare a tobacco angular leaf spot suspension (1×10 5 CFU / mL). Take 200 μL of tobacco angular leaf spot suspension and add the compositions provided in Examples 1-7 and Comparative Examples 1-9 or the preparation provided in Comparative Example 10 (final concentration 9 ppb), respectively. At the same time, single nano-titanium dioxide, nano-copper hydroxide and nano-silver are used for comparison. The culture is shaken at 28°C for 12 hours. The OD values of tobacco angular leaf spot in different treatment groups before and after culture are calculated. 600 The inhibitory effect on tobacco angular leaf spot pathogen was determined by the change value of

[0087] The test results are shown in Table 1. Nano-titanium dioxide, nano-copper hydroxide and nano-silver have a synergistic effect in enhancing tobacco's resistance to tobacco angular leaf spot pathogen. The three have a significant inhibitory effect on tobacco angular leaf spot pathogen together. The three have excellent synergistic effects only within a specific ratio range. Nano-titanium dioxide, nano-copper hydroxide and nano-silver are irreplaceable compared to other metal nanoparticles. Not any combination of metal nanoparticles can significantly inhibit tobacco angular leaf spot pathogen.

[0088] Table 1

[0089] Test samples Antibacterial rate (%) Example 1 46.21 Example 2 43.89 Example 3 43.67 Example 4 39.52 Example 5 39.27 Example 6 33.06 Example 7 32.68 Comparative Example 1 25.01 Comparative Example 2 24.78 Comparative Example 3 23.95 Comparative Example 4 28.55 Comparative Example 5 29.05 Comparative Example 6 29.30 Comparative Example 7 30.22 Comparative Example 8 29.56 Comparative Example 9 28.99 Comparative Example 10 23.04 Nano-titanium dioxide 22.47 Nano copper hydroxide 20.01 Nanosilver 23.01

[0090] Test Example 2

[0091] Tobacco K326 seeds were sown on nutrient soil and transplanted into individual pots after germination, when two to four true leaves appeared. When the seeds reached the six-leaf, one-heart stage, an inoculation experiment with tobacco angular leaf spot pathogen was conducted. One day before inoculation, the tobacco plants were sprayed with 10 mL of 9 ppb of the compositions provided in Examples 1-7 and Comparative Examples 1-9, or the formulation provided in Comparative Example 10. Nano-titanium dioxide, nano-copper hydroxide, and nano-silver alone were also used for comparison. A blank control group was not treated before inoculation.

[0092] Pseudomonas syringae pv. angulata (kindly provided by Zhengzhou Tobacco Science Research Institute) was cultured in LB liquid medium with shaking at 28°C overnight. The bacterial solution was centrifuged at 5000 rpm for 10 min, and the bacterial pellet was resuspended in PBS solution for washing. The pellet was centrifuged again and resuspended in PBS solution to prepare a tobacco angular leaf spot suspension (1×10 5 CFU / mL).

[0093] Puncture the leaf with a 1mL syringe needle. Press the upper surface of the leaf with one hand at the small hole and inject 200μL of bacterial solution from the lower surface of the leaf. Inoculate 10 tobacco plants in each group. Inoculate on the two fully expanded leaves at the top of each tobacco plant. The inoculation position is random. 1-10 days after inoculation, observe and record the number of lesions on the leaves. If necrotic spots appear on the leaves and there is a surrounding yellow halo, the leaves are judged to be infected if typical symptoms of tobacco angular leaf spot appear. Take pictures on the 10th day and count the area of lesions ( Figure 1 ).

[0094] The test results are shown in Table 2. Nano-titanium dioxide, nano-copper hydroxide and nano-silver have a synergistic effect in enhancing tobacco's resistance to tobacco angular leaf spot pathogens. The three work together to have a significant preventive and therapeutic effect on tobacco angular leaf spot. The three have excellent synergistic effects only within a specific ratio range. Nano-titanium dioxide, nano-copper hydroxide and nano-silver are irreplaceable compared to other metal nanoparticles. Not any combination of metal nanoparticles can significantly prevent and treat tobacco angular leaf spot.

[0095] Table 2

[0096]

[0097]

[0098] The present invention uses the above-described embodiments to illustrate the use of a composition for preventing and treating tobacco angular leaf spot in enhancing tobacco resistance to tobacco angular leaf spot pathogens. However, the present invention is not limited to these embodiments, and implementation of the present invention is not necessarily dependent on these embodiments. Those skilled in the art will appreciate that any improvements to the present invention, equivalent substitutions for various raw materials in the product of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0099] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0100] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A composition for preventing and treating tobacco angular leaf spot in enhancing tobacco resistance to tobacco angular leaf spot pathogen, characterized in that: The composition comprises nano titanium dioxide, nano copper hydroxide and nano silver.

2. The use according to claim 1, characterized in that The composition comprises 30-55 parts of nano titanium dioxide, 10-34 parts of nano copper hydroxide and 5-20 parts of nano silver in parts by weight.

3. The use according to claim 1 or 2, characterized in that The composition comprises 35-45 parts of nano titanium dioxide, 15-25 parts of nano copper hydroxide and 10-15 parts of nano silver in parts by weight.

4. A fungicide for preventing and treating tobacco angular spot disease, characterized in that: The bactericide comprises a composition for preventing and treating tobacco angular spot disease and an agriculturally acceptable functional adjuvant; the composition comprises nano titanium dioxide, nano copper hydroxide and nano silver.

5. The bactericide according to claim 4, characterized in that The composition comprises, by weight, 30-55 parts of nano titanium dioxide, 10-34 parts of nano copper hydroxide and 5-20 parts of nano silver; Preferably, the composition comprises 35-45 parts of nano titanium dioxide, 15-25 parts of nano copper hydroxide and 10-15 parts of nano silver in parts by weight.

6. The bactericide according to claim 4 or 5, characterized in that The mass percentage of the composition in the fungicide is 10-95%; Preferably, the mass percentage of the composition in the fungicide is 22-86%.

7. The bactericide according to any one of claims 4 to 6, characterized in that The agriculturally acceptable functional adjuvant includes any one or a combination of at least two of solvents, emulsifiers, dispersants, wetting agents, adhesives, stabilizers, defoaming agents, diluents, thickeners or antifreeze agents.

8. The bactericide according to any one of claims 4 to 7, characterized in that The dosage form of the bactericide includes any one of powder, suspension, microemulsion, ointment, plaster or smear; Preferably, the fungicide is applied by any one of foliar spraying, soil mixing, root irrigation, seed soaking, furrow application or hole application, or a combination of at least two of them.

9. A method for preventing and treating tobacco angular spot disease, characterized in that: The fungicide according to any one of claims 4 to 8 is applied to tobacco and / or its cultivation medium before or after tobacco is infected.

10. The method according to claim 9, characterized in that Nano titanium dioxide, nano copper hydroxide and nano silver are applied simultaneously or sequentially.

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