阅读说明：本技术 硫代丁酸甲酯在防治番茄枯萎病中的应用 (Application of methyl thiobutyrate in preventing and treating tomato wilt ) 是由 刘锐 赵建龙 茆振川 凌键 李彦 赵松宇 杨宇红 谢丙炎 于 2021-11-04 设计创作，主要内容包括：本发明提供了硫代丁酸甲酯在防治番茄枯萎病中的应用,及一种用于防治番茄枯萎病的组合物,所述组合物包含硫代丁酸甲酯。本发明首次提出了硫代丁酸甲酯在防治番茄枯萎病中的应用,硫代丁酸甲酯是生物天然产物,对人类及环境无残留危害,安全性较高。本发明实验证实硫代丁酸甲酯溶液在浓度大于等于5mg/ml可以几乎100％的杀死番茄枯萎菌,具有显著的效果；在盆栽试验中,采用2mg/ml的ME溶液处理对番茄枯萎的防治效果可以达到67.4％。本发明的组合物对番茄枯萎病表现出优良的防治效果。(The invention provides an application of methyl thiobutyrate in preventing and treating tomato blight and a composition for preventing and treating tomato blight, wherein the composition comprises methyl thiobutyrate. The invention provides the application of methyl thiobutyrate in preventing and treating the tomato wilt for the first time, wherein the methyl thiobutyrate is a biological natural product, has no residue harm to human and environment, and has higher safety. Experiments prove that the methyl thiobutyrate solution can almost 100 percent kill tomato fusarium wilt bacteria at the concentration of more than or equal to 5mg/ml, and has remarkable effect; in a pot experiment, the control effect of the treatment of 2mg/ml ME solution on tomato withering can reach 67.4 percent. The composition of the invention shows excellent control effect on tomato wilt.)

1. Application of methyl thiobutyrate in preventing and treating tomato wilt.

2. A composition for controlling tomato wilt disease, said composition comprising methyl thiobutyrate.

3. The composition for controlling tomato wilt disease according to claim 2, characterized in that said composition further comprises absolute ethanol, wherein the mass ratio of methyl thiobutyrate to absolute ethanol is 1: 1 to 5.

4. The composition for controlling tomato wilt disease according to claim 3, wherein the mass ratio of methyl thiobutyrate to absolute ethyl alcohol is 1: 2.

5. application of methyl thiobutyrate in regulation and control of expression of tomato fusarium wilt-related genes.

6. Use of the composition of any one of claims 2 to 4 for modulating the expression of tomato fusarium wilt-related genes.

7. The use according to claim 5 or 6, wherein the tomato fusarium wilt-related gene is selected from at least one of the group consisting of Actin, serP, FolStuA, FolCTS3 and SIX gene.

8. The use according to claim 5 or 6, wherein the regulation of expression of the tomato fusarium wilt-related gene is reduction of expression level of the tomato fusarium wilt-related gene.

9. The use according to claim 5 or 6, wherein the expression of the tomato fusarium wilt bacterium-related gene is the expression of the mRNA level of the tomato fusarium wilt bacterium-related gene or the expression amount of the protein level of the tomato fusarium wilt bacterium-related gene.

10. A method for controlling tomato wilt, characterized in that a composition according to any one of claims 2-4 is used.

Technical Field

The invention relates to an application of methyl thiobutyrate in preventing and controlling tomato blight and a composition for preventing and controlling tomato blight, belonging to the technical field of agricultural disease prevention and control.

Background

Tomato blight, also known as wilting disease, is a soil-borne disease caused by Fusarium oxysporum tomato specialization (Fusarium oxysproum f.sp.lycopersici (Sacc.) Snyder & Hansen) which is a deuteromycete tomato specialization fungus. The wilt is an important disease worldwide and has wide distribution range, the disease incidence of common diseased plants is 3-5 percent and can seriously reach more than 10 percent, even the whole plants die. Huge losses are caused to tomato production every year. The wilt disease is a typical soil-borne disease of solanaceous vegetables, causes serious continuous cropping obstacle, mainly invades from a wound at the root or stem base of a host, propagates in a vascular bundle after invasion, spreads and expands upwards to block the vascular bundle, influences water transportation, causes plant wilting death, and simultaneously generates toxin during infection to cause plant cell necrosis and vascular bundle necrosis.

Tomato blight usually occurs after flowering, the middle and lower leaves of the plant at the early stage of disease wilt in the middle of the day, and return to normal in the morning and evening, more leaves in the middle and upper part of the plant begin to wilt with the development of disease, and finally the whole plant withers due to leaf withering and yellowing of the whole plant. In the middle stage of disease development, water-soaked yellow brown to dark brown necrotic streak spots appear on one side of the stem base of the plant. When the air is moist, a little pink mildew layer appears at the diseased part, the diseased plant is pulled out, the diseased stem is longitudinally cut, and the vascular bundle is discolored. The strain can live through the overwintering of mycelium or chlamydospore in soil and can also carry out saprophytic life, seeds can carry pathogenic bacteria and the like, the control of tomato blight is very difficult, and the yield and the quality of tomatoes are seriously influenced once the tomato blight occurs.

The main methods for preventing and treating the disease at present are as follows: biological control, agricultural control and the like. In the prevention and treatment process, a large amount of chemical pesticide is used for treatment, so that the problems of environmental pollution, pesticide residue and the like are caused, and the healthy development of human beings is seriously harmed. In recent years, along with the increasing attention on the safety problem of agricultural products, the safety concept of pesticides is changed, and a novel safe and efficient medicament is urgently required to be found for preventing and treating blight. Under the background of structural improvement of the national agricultural supply side, the antagonistic microorganism and the metabolite thereof become hot points for preventing and treating soil diseases, and have become the first choice for preventing and treating soil-borne diseases due to the advantages of convenient culture, low production cost, ecological safety, strong specificity and the like.

Disclosure of Invention

The invention aims to provide an application of methyl thiobutyrate in preventing and controlling tomato blight and a composition for preventing and controlling tomato blight, wherein the methyl thiobutyrate directly kills pathogenic bacteria, so that the effect of preventing and controlling tomato blight is realized; the composition is derived from natural products of biocontrol bacteria, has the characteristics of safety, high efficiency and low residue, and is suitable for preventing and controlling the tomato wilt; the problem of serious environmental pollution caused by pesticide residues is solved while the tomato wilt is safely and efficiently prevented and controlled, and green prevention and control for the tomato wilt is realized.

According to one aspect of the present application, there is provided the use of methyl thiobutyrate for the control of tomato wilt disease.

According to another aspect of the present application, there is provided a composition for controlling tomato wilt disease, said composition comprising methyl thiobutyrate.

In some embodiments, the composition further comprises absolute ethanol, wherein the mass ratio of methyl thiobutyrate to absolute ethanol is 1: 1-5; preferably, the mass ratio of the methyl thiobutyrate to the absolute ethyl alcohol is 1: 2.

in some embodiments, the composition is formulated by: weighing 1000 mu g of methyl butyrate liquid and 2000 mu g of absolute ethyl alcohol liquid, uniformly mixing, transferring to a 100mL brown volumetric flask, metering volume with clear water, and uniformly shaking for use. Because methyl thiobutyrate and absolute ethyl alcohol have volatility, the prepared solution needs to be prepared at present, and volatilization degradation is avoided.

According to another aspect of the present application, there is provided the use of methyl thiobutyrate for modulating the expression of a gene associated with tomato wilt bacteria.

According to another aspect of the present application, there is provided the use of said composition for regulating the expression of tomato fusarium wilt-related genes.

In some embodiments, the tomato fusarium wilt-related gene is selected from at least one of the group consisting of Actin, SerP, FolStuA, FolCTS3, and SIX genes.

In some embodiments, the modulating expression of a tomato fusarium wilt cell-related gene is decreasing expression of a tomato fusarium wilt cell-related gene.

In some embodiments, the expression of the tomato fusarium wilt bacterium-related gene is the expression of the mRNA level of the tomato fusarium wilt bacterium-related gene or the expression amount of the protein level of the tomato fusarium wilt bacterium-related gene.

According to another aspect of the present application, there is provided a method for controlling tomato blight using the above composition. The composition comprises methyl thiobutyrate and absolute ethyl alcohol, wherein the mass ratio of the methyl thiobutyrate to the absolute ethyl alcohol is 1: 1-5; preferably, the mass ratio of the methyl butyrate to the absolute ethyl alcohol is 1: 2.

the invention has the beneficial effects that:

1. the invention provides the application of methyl thiobutyrate in preventing and treating tomato wilt for the first time, wherein the methyl thiobutyrate and the absolute ethyl alcohol are biological natural products, are derived from biological metabolic products, have no residual harm to human beings and the environment, and have higher safety.

2. According to the composition containing methyl thiobutyrate, provided by the invention, a detection experiment on a culture medium plate proves that a methyl thiobutyrate solution can kill tomato fusarium wilt bacteria almost 100% at a concentration of more than or equal to 5mg/ml, and has a remarkable effect; in a pot experiment, the control effect of the treatment of 2mg/ml ME solution on tomato withering can reach 67.4 percent. The composition shows excellent control effect on tomato wilt in culture medium plate detection and pot experiment.

3. The composition has excellent prevention and control effects on the tomato wilt, and the methyl thiobutyrate and the absolute ethyl alcohol are biological natural products, are derived from biological metabolic products, have no residual harm to human beings and the environment, and are beneficial to realizing green prevention and control of the tomato wilt; meanwhile, pollution-free production of vegetables is guaranteed, the problems of excessive pesticide residue and the like are solved, and the method has great application potential and is suitable for agricultural development requirements.

Drawings

FIG. 1 shows the effect of different concentrations of ME solutions on the inhibition of Fusarium oxysporum; wherein, A is clear water control, B is ME solution treatment of 0.1mg/ml, and C is ME solution treatment of 5mg/ml

FIG. 2 shows the effect of killing wilt disease by ME solutions of different concentrations, wherein A is clear water control, B is ME solution treatment of 0.1mg/ml, C is ME solution treatment of 1mg/ml, and D is ME solution treatment of 5 mg/ml.

FIG. 3 shows the qPCR detection result of the gene related to tomato wilt disease by ME solution treatment.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the following examples of the invention, methyl thiobutyrate (C)₅H₁₀O_S) Purchased from chemical substances, purchased from chemical Limited of Huaweiruike, Beijing, a Jiuding chemical company, with a purity of 98% and a MW of 118.20. Appearance and properties: the transparent yellow liquid has stimulating effect. Appearance and properties: clear yellow liquid, slightly soluble in water, density: 0.966g/mL at 25 ℃, boiling point: 142 ℃ and 143 ℃, 757mm Hg, refractive index: n 20/D1.461, vapor pressure: 5.87mmHg 25 ℃.

Example 1 inhibition of Fusarium oxysporum by methyl thiobutyrate

1. Isolation and purification of pathogenic bacteria

1.1PDA Medium

200g of peeled potato, 20g of glucose and 20g of agar powder, and the volume is fixed to 1L.

1.2 preparation of methyl Thiobutyrate solution (ME)

Methyl thiobutyrate and absolute ethyl alcohol were mixed according to a ratio of 1:2 to prepare mother liquor, and then preparing the mother liquor into corresponding concentration by adopting distilled water according to the requirement.

1.2 isolation and identification of tomato Fusarium oxysporum

Tomato wilt pathogen (Fusarium oxysproum f.sp. lycopersici) was isolated from brown vascular bundle tissue of diseased tomato stems by conventional tissue isolation. The specific operation comprises the following steps: taking a little tissue from the boundary of the disease and the health, sterilizing with 5% sodium hypochlorite for 1min, transferring to 75% alcohol for 1min, and rinsing in sterile water for 3 times. As for PDA medium. Culturing at 25 deg.C for 2-3 days, extracting DNA of strain, and culturing with sp13f (GTCAGTCCATTGGCTCTCTC) and sp13r (TCCTTGACACCATCACAGAG), and sp23f (CCTCTTGTCTTTGTCTCACGA) and

two pairs of sp23r (GCAACAGGTCGTGGGGAAA) primers are used for PCR detection to identify the variety of the Fusarium wilt, and through PCR identification analysis, a 445pb band can be obtained from the sp13f/r primer pair of the strain, and the homology with a specific sequence of the tomato Fusarium oxysporum f.sp.Lycopersici, ID: XM-018395501.1 reaches 100 percent through comparison. The method adopts sp23f/r to be incapable of amplifying a band, combines the amplification results of two pairs of primers and a comparison result to identify that the Fusarium oxysporum is a tomato Fusarium oxysporum f.sp.lycopersici No. 1 physiological race strain. The identified tomato wilt treatment strains are transferred to a PDA inclined plane and stored in a refrigerator at 4 ℃ for test analysis.

1.3 bacteriostatic experiments

Adding ME mother liquor into melted PDA culture medium when the temperature is reduced to about 40 ℃ to ensure that the final concentration of ME in the PDA culture medium is 10mg/ml, 5mg/ml, 2mg/ml, 1mg/ml and 0.1mg/ml (based on the amount of methyl thiobutyrate), pouring the mixture into a culture dish (9 x 9cm), placing the culture dish upside down and rapidly cooling to prevent the ME solution from volatilizing. A bacterial cake (0.5cm) for culturing tomato fusarium wilt for about one week is placed in the center of a culture dish by using a sterile puncher, the tomato fusarium wilt is cultured in an incubator at 28 ℃ in a dark place, the diameter of the fusarium wilt is measured after 7 days, and the bacteriostasis rate and the correction bacteriostasis rate are calculated. The test was repeated 3 times with 3 dishes of each treatment, using clear water and absolute ethanol (10mg/ml) as controls.

The growth conditions of the fusarium wilt are different under different concentrations of ME, but the growth speed of hyphae is reduced along with the increase of the concentration, the fusarium wilt hardly grows in ME solutions of 10mg/ml and 5mg/ml, the bacteriostasis effect reaches 100 percent, and the ME compound has obvious inhibition effect on the fusarium wilt of tomatoes (figure 1). In the treatment of 2mg/ml, the correction inhibition effect of the ME solution on the fusarium wilt reaches 93.3%, while in the treatment of 0.1% mg/ml, the correction inhibition effect is only 20.1%, which shows that positive correlation exists between the inhibition effect and the concentration. The specific data are set forth in Table 1 below.

The formula for calculating the bacteriostatic effect is as follows:

TABLE 1 bacteriostatic effect of different concentrations of the agents (colony diameter cm)

Example 2 killing of wilt disease by methyl Thiobutyrate

Taking methyl thiobutyrate liquid ME by a liquid transfer machine, putting the methyl thiobutyrate liquid ME into a 1.5ml centrifuge tube, mixing the liquid ME with absolute ethyl alcohol according to the proportion of 1:2, uniformly shaking, preparing solutions of 0.1mg/ml, 1mg/ml, 2mg/ml, 5mg/ml and 10mg/ml by using distilled water respectively according to the quantity of methyl thiobutyrate, and uniformly shaking for use. Because methyl thiobutyrate and absolute ethyl alcohol have volatility, the prepared solution needs to be prepared at present, and volatilization degradation is avoided.

Activating and culturing tomato fusarium wilt on PDA culture medium, then shake culturing in liquid PD culture (150rpm), and after culturing for 7 days, adopting sterilizedFiltering with double-layer gauze to remove hypha, retaining only pathogenic spore to form spore suspension, detecting spore concentration under microscope, sucking 50ul suspension, transferring into 1.5ml centrifuge tube, adjusting spore concentration with sterile water to about 1 × 10⁷cfu/ml. Adding 10 mul of bacterial liquid into 1ml of ME solution prepared with different concentrations, shaking uniformly, treating for 5h, then sucking 20 mul of bacterial liquid respectively, coating on a PDA plate, culturing at 28 ℃ in a dark place, and detecting the number of bacterial colonies on the plate in 3-7 days. The test was carried out with clear water and absolute ethanol (10mg/ml) as controls. Each treatment had 3 dishes and the experiment was repeated 3 times.

After 3 days of culture, the growth and number of bacterial colonies on the plate are detected, and the bacterial colonies of the fusarium wilt bacteria grow to fill the culture dish in the clear water control, and the number of thalli on each PDA plate reaches 6.7 multiplied by 10²cfu, which is connected into a whole on the whole culture dish, has no obvious difference from clear water in the growth condition of the bacterial colony in the absolute ethyl alcohol (10mg/ml) treatment, and is connected into a whole. No colonies of Fusarium oxysporum grew out of the ME solutions at 10mg/ml and 5mg/ml, indicating that the ME solutions were effective in killing Fusarium oxysporum at these concentrations. In the combined solution of 1mg/ml, only a few colonies are present, the average number is 4.7 colonies per dish, and the sterilization effect reaches 99.3%. Approximately 27.3 colonies per dish in a 0.1mg/ml treatment gave a bactericidal effect of 95.9%. The test proves that the ME solution has good prevention and control effects on the pathogenic bacteria of the wilt when the ME solution is more than 1 mg/ml. See table 2 below and fig. 2 in particular.

The calculation formula of the sterilization effect is as follows:

TABLE 2 Sterilization effect (cfu/dish) for different concentrations treatment

Example 3 analysis of the inhibitory Effect of ME on the expression of the wilt disease Gene

Taking methyl thiobutyrate liquid by a liquid transfer machine, putting the methyl thiobutyrate liquid into a 1.5ml centrifugal tube, mixing the liquid with absolute ethyl alcohol according to a mass ratio of 1:2, uniformly shaking, preparing 2mg/ml solution by using distilled water respectively according to the mass of the methyl thiobutyrate as a standard, and uniformly shaking for use. Because methyl thiobutyrate and absolute ethyl alcohol have volatility, the prepared solution needs to be prepared at present, and volatilization degradation is avoided.

Activating and culturing tomato fusarium wilt on a PDA culture medium, culturing for 5 days on a solid PDA culture medium, lightly scraping 50mg of thalli (containing hyphae and spores) on the surface of the PDA by using a sterilized scalpel, putting the thalli into a 2.0ml centrifuge tube, adding 1.5ml of 2mg/ml ME solution, soaking for 4 hours, centrifuging for 5 minutes at 4 ℃ by using a centrifuge 12000rpm, removing supernatant, retaining the thalli, performing suspension washing for 2 times by using sterile water, sufficiently removing residual ME solution, centrifuging for 5 minutes at 4 ℃ by using 12000rpm again, removing the supernatant, and extracting total RNA of a sample from the precipitated thalli by using a total RNA extraction kit of Tiangen biochemistry (TIANGEN). cDNA was obtained using TAKARA's reverse transcription kit (primescript RT reagent kit) and made up to 50. mu.l. Samples (1. mu.l) were each taken and subjected to expression level analysis of the target gene, and the gene expression level analysis was carried out using a qPCR kit for TAKARA (premix EX Tag II), and the absolute quantitative analysis (Delta CT method) was used for the gene expression analysis. The test was run with a clear water mock treatment. The experiment was repeated 3 times. Differential analysis of Gene expression relative expression analysis with clear water control and ME treatment (1: 2)^-△CT). The names, characteristics and primer information of the genes detected are shown in Table 3, wherein actin (actin) is an active gene representing tomato blight germ, serine protease (SerP). In addition, the related pathogenic genes of the tomato fusarium wilt pathogen are detected, including chitinase gene FolCTS3 of the fusarium wilt pathogen, pathogenic related transcription factor gene FolStuA and secretory protein gene SIX1-4 of the fusarium wilt pathogen in tomato xylem.

The qPCR results (FIG. 3) show that after 2mg/ml ME solution is soaked for 4h, the detection genes of tomato fusarium wilt bacteria show a remarkable reduction phenomenon (Table 4), and the active gene Actin and the serine protease gene SerP are remarkably reduced by 7.16 times and 4.38 times respectively. Meanwhile, qPCR detection of tomato fusarium wilt bacteria also proves that pathogenic related genes are remarkably reduced, wherein pathogenic related transcription factor gene FolStuA is reduced by 43.11 times, and chitinase gene FolCTS3 is reduced by 10.48 times. The qPCR detection result shows that the wilt disease xylem secretory protein gene SIX has great difference with the active gene Actin under the normal condition, the CT value of the Actin is 20.01, and the CT values of the wilt disease xylem secretory protein gene SIX are all larger than 31, which proves that the expression level of the wilt disease secretory protein gene SIX under the in vitro condition is very low, and simultaneously, the great reduction degrees of different gene tables after the ME solution treatment also have difference, wherein the SIX1 is reduced by 4.32 times, and the SIX2-4 is not significantly reduced (less than 2 times). The gene expression analysis further shows that the ME solution has good inhibition effect on the activity and pathogenicity of the blight pathogenic bacteria.

TABLE 3 fluorescent quantitative detection primers

TABLE 4 fluorescent quantitative differential analysis of gene expression

Gene	CK control	ME treatment	△CT	2-△CT	CK：ME
						Actin	20.01	22.85	2.84	1/7.16	1:0.14
FolCTS3	28.19	31.58	3.39	1/10.48	1:0.10
						FolStuA	23.51	28.94	5.43	1/43.11	1:0.02
SerP	30.99	33.12	2.13	1/4.38	1:0.23
						SIX1	31.76	33.87	2.11	1/4.32	1:0.23
SIX2	33.27	34.07	0.8	1/1.74	1:0.57
						SIX3	32.23	32.63	0.4	1/1.32	1:0.76
SIX4	32.45	33.02	0.57	1/1.48	1:0.67

Example 4ME Pot identification test for prevention and control effect of wilt disease

Activating and culturing tomato fusarium wilt on PDA plate culture medium for 7 days, selecting a little hypha, culturing in a constant temperature shaking incubator with PL liquid culture medium at 24-26 deg.C for 2-3 days, filtering, collecting spores, and preparing into 4 × 10 with sterilized distilled water⁶The spore suspension is inoculated by using the inoculation suspension of each spore/ml.

The method comprises the steps of disinfecting tomato seeds of 'Lichun' and accelerating germination at 28 ℃, planting the tomato seeds in a seedling tray when the seeds are exposed to the white, carrying out normal management by using a culture medium which is a disinfected grass carbon and vermiculite mixture, slightly pulling up the tomato seedlings by adopting a root soaking inoculation method when the tomato seedlings have 4 true leaves, washing the tomato seedlings by using clear water, soaking the whole root systems in an inoculated spore suspension for 10min, planting the tomato seedlings in culture bowls (9 multiplied by 10cm), planting 1 seedling in each bowl, irrigating 10ml of 2mg/ml ME solution in each bowl in an ME treatment group, treating 10 plants each, and repeating for 3 times. The negative control is that 10ml of clear water is added into each pot for irrigation; meanwhile, 500 times of 80% carbendazim (Shanghai Digai) solution is set, and 10ml of the carbendazim solution is poured into each pot to serve as a medicament treatment positive control. The temperature after inoculation is 25-30 ℃, the soil humidity is 85-90%, the illumination is 12-14 h every day, and the cultivation management is normal. The investigation was carried out about 10 days after inoculation.

The disease grading standard of the tomato blight is as follows: grade 0, no symptoms; grade 1, 1-2 cotyledons obviously turn yellow and fall off; grade 2, 1-2 true leaves become yellow or the whole leaves become yellow-green, and the leaves droop slightly and wilfully; grade 3, the whole plant is obviously wilted or the leaves are seriously yellow, and the plant growth is hindered and slightly dwarfed; and 4, the whole leaf is severely wilted or even died. The disease index is counted according to the disease condition, and the prevention and treatment effect is further calculated by the following formula. As can be seen from the results in Table 5, the disease index of tomato blight was significantly reduced after treatment with 2mg/ml ME solution, and the average disease index was 27.5. The disease index of the control reached 84.5. The prevention and treatment effect of the three repeated tests reaches 67.4 percent, which is slightly lower than that of carbendazim medicament treatment by 70.4 percent, and the difference between the two is not obvious in variance analysis. The pot inoculation test result further shows that the methyl thiobutyrate and absolute ethyl alcohol combined solution has a good application effect on preventing and controlling withering of tomatoes, and provides a basis for development and application.

Calculating the disease index:

investigating the disease condition of each identification material, investigating the materials one by one according to disease symptom grading description, recording the disease grade, and calculating the disease index.

The disease index is calculated according to the following formula:

in the formula: sigma-each disease level represents the sum of the product of the numerical value and the disease level disease number corresponding to the numerical value;

s-each disease level represents a numerical value;

n-number of diseased plants at each disease level;

n-investigating total plant number;

m-highest disease index.

The prevention and treatment effect calculation formula is as follows:

TABLE 5 potted plant effect for prevention and control of tomato blight

The application of methyl thiobutyrate provided by the invention in preventing and treating tomato wilt is described in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.