CN103703121A - Novel strain of Bacillus subtilis, and use thereof for preventing root rot disease in plants - Google Patents

Abstract

The invention relates to a novel strain of Bacillus subtilis, and more particularly, to the B-5604 strain of Bacillus subtilis, which inhibits the growth of root rot pathogens, to a composition containing the strain or the culture medium thereof for controlling root rot pathogens, to a method for controlling ginseng root rot pathogens using the composition, and to a method for inhibiting the growth of Cylindrocarpon destructans or Fusarium solani using the strain. The microorganisms having antagonistic activity developed according to the present invention are not harmful to water and soil, and thus does not cause environmental pollution, and particularly, the microorganisms having antagonistic activity according to the invention may not only reduce root rot pathogens in farmland in which ginseng is repeatedly cultivated or recultivated, but also alleviate root rot disease in ginseng being cultivated, thus significantly contributing to ginseng cultivation.

Description

Novel bacillus subtilis strain and its application to plant root rot control

technical field

The present invention relates to a novel bacillus subtilis bacterial strain, more specifically to a kind of bacillus subtilis bacterial strain B-5604 which hinders the growth of root rot pathogenic bacteria, and a composition for preventing and treating root rot pathogenic bacteria comprising the above bacterial strain or its culture solution , and a method for controlling the pathogenic bacteria of ginseng root rot by using the above-mentioned composition, and a method for inhibiting the growth of rust-rot fungus or Fusarium solani by using the above-mentioned strain. the

Background technique

Before 1992, the research on root rot of plants was mainly carried out on a generalized root rot caused by Cylindrocarpon destructans (Zinssm.) Scholten, in which rust rot fungi are compound Root rot and ginseng root rot pathogens, and the research on root rot in the real sense has been concentrated since 1992. However, there are still very few research results on this pathogenic bacteria. It has been reported that the above-mentioned pathogens are found worldwide, mainly in trees or bulbs such as daffodils, cyclamen, alfalfa and ginseng (Table 1). the

Table 1 The main host range of rust fungus (Zinssm.) Scren

方式来确保耕种地是初次耕种地。但是由于可耕作人参的初次耕种地的持续减少，以及初次耕种地的绝对不足引起的远距离出张耕作 

从而导致生产费用的增加和疏忽管理，以及对新地域气象和土壤条件变化的适应不足，使人参的生产率和品质降低。收获人参后4-5年内再次连作或者轮作人参时，人参棚子内的根部会腐败，并呈黑褐色，发生叶子早期红叶化的根腐病。  Many researchers use a variety of methods to prevent and control ginseng root rot, but due to the lack of pathogenesis of root rot caused by rust fungi and basic research on the physiology and ecology of pathogenic bacteria, there is still no effective prevention or control method . Moreover, although it has been clarified that the pathogenic bacteria of root rot in the soil is the reason for hindering continuous cropping, since there is no effective control method after the onset, the root rot produced after transplanting ginseng can only be controlled at present to prevent it from spreading in the greenhouse. Peripheral diffusion. In ginseng farming, crop rotation refers to cultivating other crops for 7-10 years after harvesting ginseng, and then re-cultivating ginseng, while continuous cropping refers to not cultivating other crops after harvesting ginseng, and managing the farming site for 1-2 years Cultivate ginseng again. Ginseng is a typical crop that is hindered by continuous cropping. There is an absolute shortage of primary arable land due to the increase in the required cultivation area in major ginseng producing areas. Therefore, it is currently cultivated by transferring it to other areas, that is, through the so-called In and out farming

Ways to ensure that the cultivated land is the first cultivated land. However, due to the continuous reduction of the primary cultivated land that can be cultivated for ginseng, and the absolute shortage of primary cultivated land, long-distance farming is caused.

Thereby leading to the increase of production cost and negligent management, as well as the lack of adaptation to the change of new regional weather and soil conditions, the productivity and quality of ginseng are reduced. When ginseng is re-cropped or rotated within 4-5 years after harvesting ginseng, the roots in the ginseng shed will rot and turn dark brown, and root rot will occur in which the leaves turn red in the early stage.

Ginseng farmers believe that in the first cultivation land, the root rot pathogen density in the soil is not very high, so when harvesting 6-year-old ginseng, the proportion of extinct plants caused by early root rot is about 50%. The occurrence of disease, except for special circumstances, generally occurs in a part of the 50% ratio of spoilage disappearance in breeding. However, in the sheds where continuous cropping obstacles occur, the rate of early wilting caused by root rot within 1-2 years of transplantation is 30%, and in severe cases it can reach 80%, and it develops rapidly afterwards, and the wilting rate among 3-4 years old ginseng The rate can reach 100%. the

The symptoms of continuous cropping disorder mentioned above are that the roots are rotted into dark brown, and the leaves dry out at an early stage, and the symptoms of root erosion appear. This symptom is caused by a plant pathogenic fungus, Cylindrocarpon destructans (Zinssm. ) Scholten), after confirming this point, the physiological characteristics of the pathogenic bacteria and the amino acid availability and secretion in culture were studied. The fungus was originally named C. radiicicola, and was later renamed rust fungus by Scholten. Some researchers have reported on the difference in mycelial production and toxin production of rust fungi under different carbon and nitrogen sources in the culture medium, as well as the impact of temperature on the growth of the fungus. Reports on the incidence characteristics of root rot and the growth differences caused by various factors such as culture temperature, light, medium pH, and medium types. In terms of growth characteristics, after the isolation and confirmation of the root rot pathogen related to the continuous cropping obstacle of ginseng, that is, the rust rot fungus, due to the concern about this pathogen and as a part of the research to solve the continuous cropping obstacle of ginseng, it was selected for the development of this pathogen. Medium, it is reported that there are different growth characteristics caused by different temperature, pH and other culture conditions and adding different antibacterial substances. the

According to reports, pathogens related to root rot in ginseng cultivation include Phytop hthora cactorum, Cylindrocarpon destructans, Fusarium solani, etc. Among these pathogenic bacteria, the main damage observed from the pathogenic bacteria that act as obstacles to continuous cropping is chlamydospore-forming and rust-rot fungi that use the infectivity of the soil to cause disease. From the typical black lesion tissue of the disease, the rust rot fungus was isolated and its pathogenicity was confirmed, which brought researchers' attention to the pathogen to a climax. It has been reported that attention should be paid to root rot caused by rust-rot fungi not only in continuous-cropping cultivated land but also in primary cultivated land. the

So far, known methods for reducing the density of soil pathogenic bacteria present in soil include adding water to soil in which ginseng is grown or using soil fumigants, but these methods have the disadvantage of being cumbersome to use. In autumn, after the ginseng harvested soil was treated with organic mercury fumigant, according to the changes in soil microbial density analyzed at different times, the bacteria with a faster proliferation rate returned to the level before treatment 20 days after treatment, while The density of slow-growing fungi or actinomycetes dropped sharply after the treatment, but in the spring of the second year, that is, before and after the ginseng transplantation period, the microbial density returned to the normal level, which shows its control effect on soil harmful fungi little. In addition, root rot will occur when the root rot pathogen is inoculated 30 cm from the soil surface and ginseng is transplanted, but root rot will not occur if the inoculation is below 50 cm from the soil surface, so it is expected to be fumigated to a depth of 40 cm from the soil However, in the process of ginseng cultivation, when root rot pathogens cause root corruption, because it is in the cultivation stage of ginseng, it is difficult to implement the method of using fumigants for prevention and control. the

As mentioned above, root rot is the biggest problem in ginseng farming. In order to prevent root rot, many researchers at home and abroad have tried a variety of breeding methods and chemically synthesized pesticides for prevention and control, but the control value of the screened pesticides is low, so it is difficult to use, and some farmers abuse pesticides indiscriminately , polluted the raw material ginseng, and even polluted the water quality and soil. And in foreign countries, since the importing country of imported red ginseng products has banned pesticides as the subject of supervision, even if the pesticides approved for use in Korea are banned in the importing country, attention should be paid to the use of residual pesticides. Recently, due to worldwide concerns about the hazards caused by organic synthetic pesticides and various environmental hazards, the importance of developing environmentally friendly and low-toxicity pesticides with clear mechanisms of action has been recognized. the

Contents of the invention

The technical problem to be solved in the present invention

The inventor has paid great efforts in order to explore new fungicides that are environmentally friendly, low-toxic, and capable of preventing and treating root rot. In order to prevent root rot that occurs during continuous cropping and crop rotation of ginseng, the inventors newly developed antagonistic microorganisms obtained from microbial resources that are environmentally friendly and capable of preventing and controlling microorganisms that can induce root rot of ginseng, thus completing the present invention. The active antagonistic microorganisms developed by the present invention are friendly to water quality and soil, so they will not pollute the environment. In particular, the active antagonistic microorganisms can improve the cultivated land of ginseng continuous cropping or crop rotation, and can also improve the root rot of ginseng being cultivated, so it is very much expected For its pioneering contributions in ginseng farming. the

Technical solutions

The object of the present invention is to provide a novel Bacillus subtilis strain B-5604 (preservation number: KCTC11758BP). the

Another object of the present invention is to provide a composition for controlling root rot pathogenic bacteria, the composition comprising the above strain or its culture solution as an active ingredient. the

Another object of the present invention is to provide a method for controlling pathogenic bacteria of ginseng root rot, the method using the above composition. the

Another object of the present invention is to provide a method for inhibiting the growth of rust fungus or Fusarium solani by using the above strain. the

Another object of the present invention is to provide the use of the above strain or composition for controlling root rot pathogens. the

Beneficial effect

The antagonistic microorganisms developed by the present invention have been elucidated to have specific bactericide resistance mechanism to ginseng root rot bacterial strains, prepared and explored an active substance screening system with detectable novel sterilizing mechanism and effective sterilizing, thereby It can be used as an active microorganism with a new mechanism of action and capable of overcoming the mechanism of sterilant drug resistance. Moreover, it can effectively hinder the growth of pathogenic bacteria of ginseng root rot, and will not cause environmental pollution to ginseng cultivation. It can provide non-toxic and environmentally friendly sterilizers and stable pesticides for ginseng root rot, which will help reduce the risk of ginseng cultivation. Reduce the operating costs of farmland, reduce environmental pollution, and stably supply and export competitive high-quality ginseng products. In addition, Fusarium solani strains have been reported to cause problems not only in American ginseng but also in Japanese ginseng, so safe active microorganisms are expected to improve national competitiveness and revitalize the national economy. the

Description of drawings

Figure 1 shows antagonistic strains having inhibitory activity against rust fungi. the

Fig. 2 represents the gene rDNA sequence (SEQ ID NO.1) of the 16S rRNA of encoding Bacillus subtilis B-5604 of the present invention. the

Fig. 3 is an electron micrograph of the antagonistic microorganism of the present invention. the

Fig. 4 is a picture showing partial control results of plant root rot by antagonistic microorganisms. the

Fig. 5 is a picture showing the results obtained whether the plants were treated with antagonistic microorganisms or not. the

Detailed ways

As an embodiment of the present invention, a novel Bacillus subtilis strain B-5604 (deposit number: KCTC11758BP) is provided. the

The characteristics of the above strains are that they can inhibit the growth of root rot pathogenic bacteria, preferably inhibit the growth of ginseng root rot pathogenic bacteria, and can prevent and control ginseng root rot, thereby solving a major problem in ginseng cultivation. the

The term "ginseng root rot" in the present invention refers to a soil plant disease that occurs in more than 50% of ginseng continuous cropping, and is a disease that causes the roots to rot into dark brown and the leaves to turn red in the early stage. So far, due to the lack of basic research on the pathogenesis of ginseng root rot and the physiological and ecological aspects of pathogenic bacteria, there is no definite prevention or control method for it. As a result of the inventor's efforts to develop new prevention or control methods, he developed a new type of bacterial strain - Bacillus subtilis strain B-5604, and first clarified that this bacterial strain can inhibit one of the pathogenic bacteria of ginseng root rot, rust rot or solanum rot The growth of Fusarium spp. can prevent and control ginseng root rot. the

The above strains of the present invention have an antagonistic ability to inhibit the growth of strains that cause root rot, preferably inhibit the growth of strains that cause root rot of ginseng. The "pathogenic bacteria of ginseng root rot" in the present invention is not particularly limited, and includes any strains that can cause root rot, and specific examples thereof include Phytophthora, Rust, Fusarium solani and the like. Preferably, the pathogenic bacteria that can be inhibited by the above bacterial strains of the present invention are rust fungi or Fusarium solani. According to specific examples of the present invention, it was confirmed that the above-mentioned bacterial strains can inhibit the growth of rust rot fungus or Fusarium solani (see Examples 1 and 4). the

The term "rust rot fungus" in the present invention can be mixed with rust rot fungus (ginseng) Scholten (Cylindrocar pon destructans (Zinssm.) Scholten). The above-mentioned pathogenic bacteria are known to prevent continuous cropping of ginseng, and are known to cause soil infectious diseases by forming chlamydospores. Moreover, the above-mentioned bacterial strain was isolated from the typical black lesion tissue of root rot, and its pathogenicity was confirmed. Therefore, if the growth of the above-mentioned pathogenic bacteria can be inhibited, ginseng root rot can be prevented and controlled. the

The term "Fusarium solani" in the present invention is the pathogenic bacterium of ginseng root rot. If it is infected by the above-mentioned fungus, the surface of the ginseng root will become light brown or brown, and soft rot will occur to the inside of the root. the

When the present inventors searched for strains of Fusarium solani and Rust rot fungus strains, that is, strains with inhibitory activity against ginseng root rot pathogens, they isolated antagonistic strains that induced mutations by exposure to ultraviolet rays, specifically from South Chungcheong Province obtained from soil samples isolated from Yincheng (Fig. 1). the

The sequence of the gene (rDNA) encoding the 16S rRNA of the bacterial strain provided by the present invention is recorded as SEQ ID NO.1 (Figure 2). Compared with the rDNA of known bacterial strains, the rDNA sequence recorded as SEQ ID NO.1 has 99% similarity to the known Bacillus subtilis strain AB017589, and 99% similarity to another known Bacillus subtilis strain AB017590 consistency. The size of the general Bacillus subtilis is 1.2-2.5 μm, and the average size of the bacterial strain provided by the present invention is 0.6-0.7×1.5-1.6 μm, which is slightly smaller than the general Bacillus subtilis, and has a flagella with a size of about It is 7.5 μm, which is about 5 times the size of the bacteria (Figure 3). By comparing the results of the classification positions of the above-mentioned strains, it is classified as Bacillus subtilis, and based on the characteristics such as fatty acid composition and ratio, thalline appearance, the bacterial strain of the present invention is named "Bacillus subtilis B-5604", And it was preserved in the Biological Resource Center of the Korea Institute of Biotechnology (111 Science Road, Yucheng District, Daejeon City, Korea) on September 03, 2010, and its preservation number is KCTC11758BP. the

According to the examples of the present invention, it was confirmed that the above strains of the present invention can inhibit the growth of Fusarium solani and rust rot (see Example 4). And, in order to analyze whether the strain of the present invention can inhibit ginseng root rot, 3/4 of the roots of ginseng were covered with soil containing root rot pathogenic bacteria, and the upper part was covered with soil that would be used as root rot. The above-mentioned strains of antagonistic microorganisms for disease control are adsorbed with vermiculite and then dried in the soil. Then the result of observation is that the part of the soil containing the root rot pathogenic bacteria has rotted, but the part containing the above-mentioned bacterial strains adsorbed on vermiculite etc., that is, the drug-attached part of the root rot prevention and control antagonistic microorganisms, the root rot is obtained. improved and showed a good growth state (Figure 4). At the same time, the cultivated land containing the pathogenic bacteria of root rot was used as a control area, and the control microorganisms of the pathogenic bacteria of root rot were adsorbed on the same amount of vermiculite, and the dried vermiculite and the soil containing the pathogenic bacteria of root rot were mixed at a ratio of 1:1. Cover the ginseng after the ratio is mixed, and use it as the experimental area. The result of comparing the control area and the experimental area is that the soil containing root rot pathogenic bacteria has more corruption, but the soil treated with the above-mentioned antagonistic microorganisms of the present invention In the group, the root rot was improved and showed a good growth state. These results mean that the bacterial strain developed by the present invention can effectively control ginseng root rot. The novel Bacillus subtilis strain B-5604 of the present invention has excellent antagonistic ability to the pathogenic bacteria of ginseng root rot, does not pollute the environment, and has no toxicity to the human body. Microorganisms of pathogenic bacteria. the

As another embodiment of the present invention, the present invention provides a composition for controlling root rot pathogenic bacteria, the composition comprising Bacillus subtilis strain B-5604 (preservation number: KCTC11758BP) or its culture solution as an effective Element. the

Root rot pathogens are as described above. the

The composition of the present invention can be prepared in the form of wettable powder, granule or capsule for the purpose of stable formulation. the

For the composition of the present invention, the above-mentioned microorganisms or culture solution can be included in the composition for preventing and treating pathogenic bacteria of ginseng root rot; for long-term storage, they can also be stored separately and mixed before use. In order to preserve the above-mentioned microorganisms for a long time, they can be stored in glycerol storage solution at a temperature below -70°C when provided separately; or frozen at -20°C to -80°C for use. the

壬基酚聚氧乙烯醚、磺化甲醛萘基钠、曲拉通100及吐温80中的一种以上。 增重剂/营养剂可以选自大豆粉、大米、小麦、黄土、硅藻土、糊精（dextrin）、葡萄糖以及淀粉中的一种以上。  The wettable powder of the present invention is prepared by drying the solid culture medium inoculated with microorganisms, pulverizing, adding surfactants and weight-increasing agents/nutrients and mixing. The above-mentioned surfactant can be selected from polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkylarylsulfonate, polyoxyethylene Alkylphenyl ether (polyoxyethylene alkylphenyl ether), sodium tripolyphosphate, polyoxyethylene alkylaryl phosphoric ester, polyoxyethylene alkylaryl ether, polyoxyethylene alkylaryl polymer, polyethylene glycol alkanes Phenyl ether

More than one of nonylphenol polyoxyethylene ether, sodium naphthyl sulfonate formaldehyde, Triton 100 and Tween 80. The weighting agent/nutrient may be selected from one or more of soybean flour, rice, wheat, loess, diatomaceous earth, dextrin, glucose and starch.

Moreover, the granules of the present invention are prepared by drying the solid culture medium inoculated with microorganisms, pulverizing them, adding surfactants, weight-increasing agents/nutrients and disintegrating agents and mixing them. Above-mentioned surfactant can be selected from polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkylsulfosuccinate, sodium alkylarylsulfonate, polyoxyethylene alkylphenyl ether , sodium tripolyphosphate, polyoxyethylene alkyl aryl phosphate, polyoxyethylene alkyl aryl ether, polyoxyethylene alkyl aryl polymer, polyethylene glycol alkyl phenyl ether, nonylphenol polyoxygen One or more of vinyl ether, sodium naphthyl sulfonate formaldehyde, Triton 100 and Tween 80. The weighting agent/nutritional agent can be selected from more than one of soybean flour, rice, wheat, loess, diatomaceous earth, dextrin, glucose and starch. The disintegrant may be selected from one or more of bentonite, talc, dialite, kaolin and calcium carbonate. the

And, the granule of the present invention can be added in microorganisms One or more of attractant, encapsulating agent, binding agent, emulsifier, dye, UV protection agent, buffer and leveling agent are prepared.

The above composition can be applied to the roots of plants or the soil around them, so as to control root rot pathogens. It is preferably applied to the root of ginseng or the soil around it to prevent and control the pathogenic bacteria of ginseng root rot. The method of applying the composition includes directly spreading (spreading), spraying (spraying), soaking (soaking) the culture fluid or strains of the above-mentioned strains, or after absorbing the culture fluid or strains of the above-mentioned strains on vermiculite, diatomaceous earth, or perlite For spreading, in the process of managing cultivated land, the method of mixing and adsorbing strain culture fluid or adsorbing strains during cultivating the land can be used. In addition, known pesticide application methods, microorganism application methods, and the like can also be utilized. the

When applying the above composition, Bacillus subtilis B-5604 can be concentratedly sprayed on the roots of ginseng or in the soil ^close to it, and the effective amount of microorganisms can be 1×10 ⁷ to 5×10 ⁷ pieces, preferably 4×10 ⁷ pieces.

Also, when the microorganisms are applied, the root of ginseng may be directly sprayed with a solution composition diluted with the above-mentioned microorganisms, and the effective amount of the microorganisms contained in the composition is 1×10 ⁶ to 1×10 per milliliter (ml) volume. ⁹ , or infiltrate ginseng for 1 to 2 hours in a solution diluted to a concentration of 1×10 ⁶ to 1×10 ⁹ microorganisms per milliliter (ml).

As another embodiment of the present invention, the present invention provides a method for preventing and treating pathogenic bacteria of ginseng root rot, the method utilizing a composition for controlling pathogenic bacteria of root rot, the composition comprising Bacillus subtilis strain B-5604 (Accession number: KCTC11758BP) or its culture solution as an active ingredient. the

The control method is to spray or soak the composition concentratedly in the root of ginseng or the soil adjacent to it, so as to prevent and control the pathogenic bacteria of ginseng root rot, and the specific content is as described above. the

The above composition can inhibit the growth of the pathogenic bacteria of ginseng root rot and can prevent and control the pathogenic bacteria of ginseng root rot. The description of the pathogenic bacteria of ginseng root rot is also as above. the

As another embodiment of the present invention, the present invention provides a method for inhibiting the growth of rust fungus or Fusarium solani using Bacillus subtilis strain B-5604 (Accession No.: KCTC11758BP). the

The above-mentioned strains, growth-inhibited strains, and methods of inhibiting growth are all as described above. the

As another embodiment of the present invention, the present invention provides the use of Bacillus subtilis strain B-5604 (preservation number: KCTC11758BP) and a composition for controlling root rot pathogenic bacteria to control root rot pathogenic bacteria. The composition contains Bacillus subtilis strain B-5604 (preservation number: KCTC11758BP) or its culture solution as an active ingredient. the

The prevention and control application can be achieved by spraying or intensively immersing the above-mentioned strain or composition in the root of the plant or the soil adjacent to it, so as to realize the application of preventing and controlling the pathogenic bacteria of root rot, and the specific content is as described above. The above-mentioned plants are preferably ginseng, so that the pathogenic bacteria of ginseng root rot can be prevented and treated. the

Thus, the above bacterial strain or composition can inhibit the growth of root rot pathogenic bacteria, thereby preventing and controlling root rot pathogenic bacteria, and the explanation about root rot pathogenic bacteria is also as above. the

Detailed ways

Hereinafter, the present invention will be described in detail through examples. These Examples are only for illustrating the present invention, and the scope of the present invention is not limited by these Examples. the

Example 1: Screening for antagonistic microorganisms

In order to search for antagonistic microorganisms that inhibit the growth of pathogenic bacteria of ginseng root rot, the present inventors screened microorganisms from soil samples in Eumseong, South Chungcheong Province. The microorganisms can grow in substances containing the inhibitory activity produced by the pathogenic bacteria of root rot, and then the antagonistic microorganisms are screened out from the above microorganisms. The search for antagonistic microorganisms that inhibit the growth of pathogenic bacteria of ginseng root rot is carried out according to the following method. The soil samples were diluted with distilled water in different gradients, and then the substance with hindering activity produced by the pathogenic bacteria of root rot was smeared on the isolation medium, so as to screen and isolate candidate strains. the

The microbial isolation medium used by the inventors (yeast extract 0.1%, polypeptone 0.5%, dipotassium hydrogen phosphate 0.05%, potassium phosphate 0.05%, magnesium sulfate heptahydrate 0.02%, agar 1.5%, hydrogen ion concentration 7.2 ) after sterilization, the temperature drops to about 50°C. In order to inhibit the growth of general bacteria, mix 1ml of colistin ( colistin) solution and a solution of Nalidixic acid (Nalidixic acid) dissolved in methanol solution at a concentration of 10 mg/L, and the separation medium was used after solidification. Dilute the soil samples in different gradients in the prepared medium for isolation and smear them, and culture them at 28°C to grow and isolate the strains. The search for antagonistic microorganisms is to screen the microorganisms that can grow in the antibiotic substances containing root rot pathogens that have hindering activity. For the exploration of antagonistic microorganisms, the microorganisms that have been screened for the first time are purified and separated, and smeared on the periphery of Fusarium solani as a root rot pathogen cultured in starch sugar agar medium, so that the second screening is effective against Growth of Fusarium solani strains have inhibitory activity of antagonistic microorganisms. the

The Bacillus subtilis (B.subtilis) that had been screened for the first time was treated with 50mM caffein, and then the surviving strains were exposed to ultraviolet light again, and the mutant-induced antagonistic strains were screened out for their resistance to rust. Bacteria strains with blocking activity (Figure 1). Inoculate Bacillus subtilis, which has an inhibitory effect on rust-rot fungus, on LBG (1.0% tryptone, 0.5% yeast extract, 1.0% sodium chloride, 1.0% glucose) and culture it for 12-18 hours, and will contain 5mM caffeine, 0.1M MgSO47H2O, 0.03% Tween 80 4ml bacterial solution (A550=0.4) was placed in a glass plate. After putting it in, two short-wavelength ultraviolet lamps (10W) and one long-wavelength UV lamp adsorbed on the upper 20cm are irradiated for 10 seconds to 2 minutes, and the strains that survived under the condition of 0.1% survival rate (when analyzed for 90 seconds) are applied. On LB agar plate, cultivate to 30 ℃ and select strains. After the selected strains were cultured in LB liquid medium, they were transferred to production medium (containing 30g of sucrose, 20g of soybean powder, 5mg of MnCl ₂ 4H ₂ O, 50mg of FeSO ₄ 7H ₂ O per milliliter) and cultured for 24-48 hours. The strains with enhanced inhibitory activity compared with the parent strain were screened out from the plate of tested bacteria with high production of saprophytes.

Example 2: Identification of antagonistic microorganisms explored

Figure 2 shows the sequence (SEQ ID NO.1) of the gene (rDNA) encoding the 16SrRNA of the antagonistic microbial strain screened in Example 1 above. Compared with the known rDNA sequences of Bacillus subtilis AB017589 and AB017590, the rDNA sequence of Bacillus subtilis screened in the above Example 1 described as SEQ No. 1 has 99% similarity to the known Bacillus subtilis strain AB017589 , while 99% identical to another known Bacillus subtilis strain, AB107590. the

The morphology of the Bacillus subtilis strains screened in the above-mentioned Example 1 was confirmed with an optical microscope, and then reconfirmed with an electron microscope at a magnification of 8800 times, and the average size of the strains was 0.6-0.7×1.5-1.6 It is about μm, slightly smaller than the general Bacillus subtilis with a size of 1.2-2.5μm, has one flagellum, and its length is about 7.5μm, which is about 5 times the size of the bacteria (Figure 3). the

However, since there are many strains with a base sequence similarity of more than 99%, it is impossible to use them for identification, and it is impossible to correctly identify them by comparative analysis of fatty acid composition. Therefore, the antagonistic microbial strains were cultured in nutrient agar (nutrient agar) plate medium for 24 hours at 37°C to obtain bacterial cells, and the obtained bacterial cells were obtained according to the method of Chun and Goodfellow (Chun J. & Goodfellow M. (1955 ) Aphylogenetic analysis of the genus Nocardia with16S rRNA gene sequences.Int.J.Syst.Bacteriol.45:240-245) After the DNA was obtained, the gyrA gene was extracted using two primers p-gyrA-f (5'-CAG TCA GGA AAT GCG TAC GTC CTT-3', SEQ ID NO.2) and p-gyrA-r (5'-CAA GGT AAT GCT ATT GCT-3', SEQ ID NO.3) for PCR to amplify the gene, for PCR conditions and base sequence analysis were identified according to the method of Chun and Goodfellow. As a result, in order to confirm the classification position of the above-mentioned strains, the most widely used microbial 16S rDNA base sequence comparison data was used as the basis, and the above-mentioned new strains were classified as Bacillus subtilis. The bacterial strain screened in the above-mentioned Example 1 of the present invention was named as the novel strain "Bacillus subtilis B-5604". And it was preserved in the Biological Resource Center of the Korea Institute of Biotechnology (111 Science Road, Yucheng District, Daejeon City, Korea) on September 03, 2010, and its preservation number is KCTC11758BP. the

Embodiment 3: the culture condition of Bacillus subtilis B-5604

Antagonist microorganisms with activity of inhibiting ginseng root rot, that is, Bacillus subtilis B-5604, can be grown in LB medium (1% casein peptone, 0.5% yeast extract, 0.5% salt, after autoclaving) at 28°C , after 24 hours of shaking culture, the obtained culture solution was mixed with 20% glycerol at a ratio of 1:1, and 2ml was added to a cryogenic bottle, which was stored frozen at -70°C and used for the production and cultivation of active substances. the

0.5%、葡萄糖0.2%、硫酸铵0.1%、磷酸钾0.1%、硫酸镁七结晶水水合物0.05%、氯化钙0.1%、氯化钠0.3%、初期pH7.0）的1L的锥形瓶中，在28℃条件下以200rpm的速率振荡培养48小时。使用硅油作为泡沫消泡剂，去除培养时产生的泡沫。  In order to produce an active substance that has inhibitory activity against ginseng root rot from active microorganisms, a cryogenic flask of frozen Bacillus subtilis B-5604 was dissolved and inoculated into 100 ml of active substance production medium (soluble starch 1%, Palmamed

0.5%, glucose 0.2%, ammonium sulfate 0.1%, potassium phosphate 0.1%, magnesium sulfate heptahydrate 0.05%, calcium chloride 0.1%, sodium chloride 0.3%, initial pH 7.0) in a 1L Erlenmeyer flask , cultured at 28°C with shaking at a rate of 200rpm for 48 hours. Use silicone oil as a foam defoamer to remove foam generated during cultivation.

Example 4: The effect of inhibiting the growth of Fusarium solani or rust rot fungus strains shown by Bacillus subtilis strain B-5604

In order to confirm the fact that the Bacillus subtilis strain B-5604 inhibits the growth of the Fusarium solani or Rust rot strain, the analysis of the growth inhibitory activity on the Fusarium solani or Rust rot strain was performed in PDA medium. After cultivating Fusarium solani or rust fungus in PDA medium at 37°C for 72 hours, use a sterilized spatula (Spatula) to take 3×3 mm, place it on PDA medium, and use sterilized Take Bacillus subtilis B-5604 from the platinum ear spoon, draw a continuous line in the shape of a "one" on the side, and culture it at 28°C for 48 hours. It can be confirmed that the growth of Fusarium solani or rust rot fungus is inhibited. the

Embodiment 5: Bacillus subtilis strain B-5604 suppresses the effect of ginseng root rot

In order to confirm the inhibitory activity of active microorganisms against ginseng root rot in ginseng cultivation fields, an experiment was conducted at the cultivation field in Jeongpyeong, South Chungcheong Province. the

The start date of the experiment was May. After confirming the presence of root rot pathogens in the ginseng cultivated land that was fallow at the time of the experiment, every 5,000 grids in the cultivated land (Note: the size of a grid is 1.8×0.9m, There were more than 500 ginsengs used in the experiment) Transplanted 2-year-old ginseng, 3/4 of the ginseng root was covered with soil containing root rot pathogenic bacteria, and the following soil was covered on it. The soil in which the antagonistic microorganisms for the prevention and treatment of Pythium root rot cultivated according to the method of above-mentioned Example 3 are adsorbed on an equivalent amount of vermiculite or the like and then dried. In August of the same year, the ginseng was excavated and the improvement degree of the treatment group of antagonistic active microorganisms for the control of root rot on ginseng root rot was analyzed. the

As a result of visually judging the degree of improvement of ginseng root rot in the treatment group with active strains for controlling root rot, it was confirmed that the soil part (lower part) containing the root rot pathogenic bacteria was rotted, but in the soil containing the fungus adsorbed on vermiculite, etc. The root rot was improved in the part where the drug-attached portion of the antagonistic microorganism grew (the upper part), and the root rot was improved, showing a good growth state (Fig. 4). the

In addition, in the root rot inhibition experiment of antagonistic microorganisms, it was confirmed that the pathogenic bacteria of root rot existed in the ginseng cultivated land, and 2-year-old ginseng was transplanted in every 5,000 grids of the cultivated land, so as to use the soil in which the pathogenic bacteria of root rot existed. Covered control groups were cultivated. The experimental group is to transplant 2-year-old root ginseng, and then the antagonistic microorganisms for the prevention and treatment of Pythium root rot cultivated by the method of the above-mentioned embodiment 3 are adsorbed on an equivalent amount of vermiculite, etc., the vermiculite is dried, and then it is mixed with The soil of root rot pathogenic bacteria was mixed at a ratio of 1:1 and covered on ginseng. In August of the same year, ginseng was dug up and the degree of improvement of root rot antagonistic active microorganism treatment group on ginseng root rot was analyzed. the

As a result of judging the degree of improvement of ginseng root rot by the treatment group with active strains for preventing and controlling root rot with the naked eye, it was confirmed that more spoilage occurred in the soil containing only the pathogenic bacteria of root rot, while the vermiculite treatment which adsorbed antagonistic active microorganisms group, whose root rot was improved, showed a good growth state (Fig. 5). the

Claims (12)

1. a bacillus subtilis strain B-5604(deposit number: KCTC11758BP).

2. for preventing and treating a composition for Pathogens Causing Root Rot Disease, it is characterized in that, it comprises bacillus subtilis strain B-5604(deposit number claimed in claim 1: KCTC11758BP) or its nutrient solution as effective constituent.

3. according to claim 2ly for preventing and treating the composition of Pathogens Causing Root Rot Disease, it is characterized in that, described Pathogens Causing Root Rot Disease is ginseng Pathogens Causing Root Rot Disease.

4. according to claim 3ly for preventing and treating the composition of Pathogens Causing Root Rot Disease, it is characterized in that, described ginseng Pathogens Causing Root Rot Disease is rust rotten bacterium (Cylindrocarpon destructans) or Fusarinm solani (Fusarium solani).

5. according to claim 3ly for preventing and treating the composition of Pathogens Causing Root Rot Disease, it is characterized in that, described composition is applicable to ginseng root or its soil around.

6. according to claim 2ly for preventing and treating the composition of Pathogens Causing Root Rot Disease, it is characterized in that, described composition is wettable powder, granule or capsule.

7. according to claim 2ly for preventing and treating the composition of Pathogens Causing Root Rot Disease, it is characterized in that, described composition is in bacillus subtilis strain B-5604 or its nutrient solution, add tensio-active agent, weighting agent and nutrition agent and make.

8. according to claim 2ly for preventing and treating the composition of Pathogens Causing Root Rot Disease, it is characterized in that, the microorganism significant quantity being contained in described composition is that every milliliter of (ml) volume comprises 1 * 10 ⁶to 1 * 10 ⁹only.

9. according to claim 2ly for preventing and treating the composition of Pathogens Causing Root Rot Disease, it is characterized in that, the described microorganism significant quantity being contained in described composition is every square of (m ²) cultivated land area is dispersed with 1 * 10 ⁷to 5 * 10 ⁷only.

10. prevent and treat a method for ginseng Pathogens Causing Root Rot Disease, described method is utilized the composition described in any one in claim 2 to 9.

The method of 11. control ginseng Pathogens Causing Root Rot Diseases according to claim 10, is characterized in that, described composition is concentrated and sprayed or impregnated in the root or soil adjacent thereto of ginseng.

12. utilize the bacillus subtilis strain B-5604(deposit number described in claim 1: the method that KCTC11758BP) suppresses rust rotten bacterium (Cylindrocarpon destructans) or Fusarinm solani (Fusarium solani) growth.

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