KR100294023B1 - Bacteria for disease prevention of crops, microorganisms containing them and uses thereof - Google Patents

Abstract

In the present invention, as a strain having a plant disease control effect, antagonism microorganisms Bacillus subtilis GB-0365 (accession number: KFCC-11071) and Bacillus sp. GB-017 (accession number: KFCC-11070), which also provides growth inhibition and fruit vegetable harvesting for major causative organisms such as fungal crop diseases, such as ash fungus, buckwheat disease, stock disease, wilting and brown patches, and large patches. It provides a microbial agent containing the strain having a maintenance effect. When the microbial agent according to the present invention is used, the active ingredient produced in the culture solution contained in the microbial agent acts as a primary to inhibit the growth of mold, and secondly, the microorganisms in the microbial agent produce the active ingredient while inhabiting the application site. By inhibiting the growth of the fungus, it is possible to reduce the disease incidence of various crops.

Description

Strains for controlling diseases of crops, microbial agents containing them and uses thereof

The present invention relates to a microorganism formulation containing the same, a microorganism formulation containing the same, and an antagonist microorganism having an effect on controlling disease and fruit vegetables, freshness after harvesting by fungi isolated from soil.

Various pests on crops directly reduce yields and affect their quality. Therefore, research on the development and use of pesticides to control pests continued to develop organic synthetic pesticides and biological agents (biological pesticides or microbial pesticides).

Organic synthetic pesticides were developed in the early 1900's and are very effective as a means of protecting crops, and have played an important role in increasing crop production, stable supply, and reducing the labor force of agricultural workers. However, in recent years, especially in developed countries, social demands such as safety-oriented, health-oriented, and natural-oriented have been increasing, and organic synthetic pesticides destroy the environment and are harmful to the human body. Much attention is being focused.

Biological pesticides are pesticides used to control insects, mites, nematodes, and various phytopathogens or weeds that harm crops by directly using microorganisms or by using microorganisms. Compared to organic synthetic pesticides, these microbial pesticides are characterized by low toxicity, low impact on the ecosystem, and no drug resistance or resistance induction, while slower drug expression and difficulties in product stability and production methods. have. Thus, until recently, the share of biopesticides in the overall pesticide market was very weak compared to organic synthetic pesticides.

However, as mentioned above, research and development on low-toxic biological pesticides are continuously concentrated as a means of preventing environmental pollution caused by the abuse of organic synthetic pesticides. Biological pesticides known to date are largely classified into microbial insecticides, microbial fungicides, and microbial herbicides.

The close relationship between microorganisms and humans is old and is with civilization. Doenjang, soy sauce, sake, pickles, and red pepper paste are all made by microorganisms, and foreign cheese, yogurt, and wine are also made by microorganisms. In agriculture, microorganisms are used in various sectors. Representative agricultural techniques using microorganisms are compost. As described above, microorganisms have been closely used in human life unconsciously and empirically. This microorganism was discovered by Levenberg in 1632, and its pure culture technology was discovered by Gogh about 100 years ago.

There are many different types of microorganisms in the soil, and the number is hundreds of billions per gram of soil. However, only 0.1% of soil microorganisms are being explained by modern science, and the remaining 99.9% are little known. It may be said that antagonists are still unknown. Plants ingest nutrients from the soil, but many fertilizers fertilized in the soil are not used in the plants as they are, and are lost all by rain or the like. Soil microorganisms decompose fertilizers and organic matter used in the soil to the form that can be used in plants and accumulate in the cells. In addition to the decomposition of fertilizers, microorganisms act to fix nitrogen in the air or to extract minerals from the rock and accumulate them into cells. Nutrients accumulated in these cells are gradually provided to plants with the propagation of microorganisms without being lost by weather. When a plant drops leaves or dies, microbes grow there. As the microorganisms multiply, the plant slowly decomposes and becomes inorganic and turns into soil. This microorganism eventually dies and turns into soil. Substances that turn into soil are also absorbed as nutrients for plants and the material cycles through. If there were no microbes, the earth would have been covered with living fluids. Thus microorganisms play a big role in the circulation of matter. This circulation is eventually due to the fact that microbes ingest various things as food to survive, but microorganisms are living influencing each other. Microorganisms need to secure their own growing environment in order to survive. To this end, it creates its own growth environment by producing substances that seem to inhibit the growth of other microorganisms. This phenomenon is called sensitization (allopati) between microorganisms. The action of killing or inhibiting pathogens by competition of food, production of substances and parasitics is called "antagonism".

As such, all microorganisms affect other microorganisms in order to survive, and among them, "antagonist microorganisms" are helpful for controlling various pests.

Due to the repeated use of pesticides, many social problems have emerged due to the emergence of drug-resistant bacteria, residual toxicity of agricultural products, and environmental pollution, and the use of pesticides is gradually being restricted, and some plant diseases are controlled by conventional chemical synthetic pesticides. Is said to be impossible. In such a situation, control by antagonism microorganisms, which are pollution-free biological pesticides, is very desirable and its development is urgently required.

In recent years, with the growing interest in biological control to use microorganisms themselves or secondary metabolites produced by microorganisms as an alternative means to solve these problems and to control only the various diseases of crops and to increase the productivity of crops, this field has been developed in developed countries. It is very active. In addition, the market for biological control is expected to expand to more than 10 trillion won in the next 10 years, and the possibility is expected to increase with the development of related biotechnology. The need for an excellent microbial fungicide is steadily increasing.

Meanwhile, research on biological control using microorganisms has made great efforts to suppress plant pathogens using many kinds of bacteria and fungi since the early 1900s. Most of the biological control agents reported to date are Agrobacterium sp., Bacillus sp., Pseudomonas sp., Streptomyces sp. Bacteria, Gliocladium virens, Trichoderma sp. Development studies using molds such as Research in this area has been particularly active in recent years and is expected to maintain high growth of 15-20% annually, forming a market of about 2 trillion won by the early 2000s. In particular, in recent years, in the process of spreading regulations on the use of toxic pesticides worldwide, many advanced countries are rushing to develop biotechnology products, and new product development and registration is proceeding at a rapid pace. More than 20 species have been registered in the United States alone. Most of the registered products are for plant diseases control, especially for Fusarium, Pythium, Rhizoctonia, Sclerotinia, Phytophthora, caused by soil transmission, but these products do not yet occupy a large market.

An object of the present invention is to provide a strain which is an antagonistic microorganism capable of controlling plant diseases caused by a fungus from soil microorganisms.

Another object of the present invention is to provide a microbial agent using the strain and a method of using the same.

The present inventors have tried to find a strain that has a control effect against plant diseases from soil microorganisms, Bacillus subtilis GB-0365 (accession number: KFCC-11071) and Bacillus sp. ) GB-017 (Accession No .: KFCC-11070) was isolated, and the control effect against mold causing major diseases of crops using microbial agents containing antagonist microorganisms and control of major diseases of crops caused by molds The invention has been completed.

The microbial agent according to the present invention has a growth inhibitory effect on the microorganisms which are the main causes of fungal diseases such as gray mold disease, diarrhea disease, wearing disease, wilted disease and brown patch, large patch, and is very effective in controlling major diseases of crops caused by mold. It is an effective microorganism.

Strains contained in microbial agents belong to GRAS (Generally Recognized as Safe) strains, which are generally isolated from natural soils and are considered safe. They are similar to the main strains of traditional fermented foods. The strain of the present invention forms endogenous spores that can survive relatively long when sprayed in nature, and produce low molecular cyclic peptides as metabolites, which exhibit antimicrobial activity against mold. Such cyclic peptides include iturin and surfactin, which have been studied in Japan and Europe and America. They have high thermal stability, little toxicity, and are broad against plant pathogenic fungi. High antimicrobial activity. Therefore, the antifungal mechanism of these strains inhibits the growth of fungi by primarily acting on the active ingredient produced in the culture medium contained in the microbial preparation, and secondly, while the microorganisms in the microbial preparation live in the field of application. It reduces the incidence of plant diseases by producing and inhibiting the growth of mold.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, and the scope of the present invention is not limited to these examples.

Example 1

[Search and Screening of Antagonistic Microorganisms]

In order to isolate the microorganisms having antagonistic activity against the disclosed pathogenic microorganisms, field soils and forest soils of Gyeonggi and Gangwon-do areas were taken as seasonal samples. Antagonist isolation was carried out using a conventional triple layer flat method. Two times distilled water was added to 250 g of high-quality field soil and autoclaved, followed by filtration using gauze. After adjusting the filtrate to 1 L with distilled water, dissolve 15 g of agar and dispense 10 ml of each 30 ml test tube, and then autoclave and store in a constant temperature water bath at 50 ° C. Subsequently, 1 g of the collected soil sample is suspended in 100 ml of sterile physiological saline, and then 2-3 drops of the suspension are mixed in the test tube, mixed well, and immediately poured into a petri dish to solidify. Dispense 5 ml of agar solution on the solidified Petri dish to solidify. Then, spore suspension of phytopathogenic microorganisms (1 × 10 ⁵ cfu / ml) was prepared from the subcultured medium, and then, after adding 2-3 drops to 10 ml of potato agar medium prepared in a 50 ° C. thermostat, the mixture was mixed well. Dispensing again in Petri dishes to prepare a trilayer flat plate. The prepared plate medium was incubated in a 30 ° C. thermostat for 3-7 days, and examined the antagonism (determined by the formation of a transparent ring) against the target plant pathogenic microorganisms, and then the fine microorganisms were separated purely by a conventional method and culture medium for general bacteria culture. After culturing using (Nutrient Broth), the conventional symmetry culture and the diameter of the transparent ring were investigated, and the highest antagonistic microorganisms GB-0365 and GB-017 were selected.

Example 2

[Identification of Antagonistic Microorganisms on Fungi That Cause Major Diseases of Crops]

When the isolated strain GB-0365 was observed by electron microscopy, it was in the form of bacilli, and it was confirmed that it was Gram-positive by gram staining and had motility. The morphological and biochemical properties of the soil-derived antagonistic microorganisms were investigated according to Bergy's Mannual, and the results are shown in Table 1 below.

This strain was identified as Bacillus subtilis, named Bacillus subtilis GB-0365, and was deposited with the Korean bacterium association as accession number KFCC-11071 as of December 4, 1998.

In addition, the isolated strain GB-017 had the form of bacilli when observed under an electron microscope, was Gram-positive, and had motility. The morphological and biochemical properties of the antagonist GB-017 according to Buggy's manual were investigated and the results are shown in Table 2 above. This strain was identified as Bacillus sp., Named as Bacillus GB-017, and was deposited with the Accession No. KFCC-1107 dated December 4, 1998 to the Korean spawn association.

Example 3

[Growth inhibition effect on various plant pathogens]

Phytopathogens include the most damaging pathogens, Botrytis cineria, fusarium sp., Pythium sp., Phytophtora capsici, and Black fungus. Ten species of Alternaria citri, Cladosporium cucumerium, anthrax (Colletotrichum gloeosporiodes), spotted disease (Pleospora sp.), Brown patch (Rhizoctonia solani MAFF 511103) and large patch (Rhizoctonia solani MAFF 305245) were used. After sterilizing the potato agar medium, plate the mycelium, cells, and spores of each pathogen in 50 ℃ medium, and after the medium is completely hardened, the paper disk (diameter 8mm) is immersed in the culture medium in which the antagonist microorganism is cultured, Placed in a dish and incubated for 3 to 4 days at a temperature well grown various pathogens. The effect of antagonistic microorganisms on various pathogens was investigated with a low support diameter. The results are shown in Table 3 below.

Referring to Table 3, as a result of antagonistic effect test of Bacillus subtilis GB-0365 strain and plant pathogen, it showed growth inhibition effect against Botrytis cineria, which causes gray mold, and inhibited mycelial growth. Symptoms were high, Fusarium sp., A cause of crop wilting disease, Pythium sp., A causative agent of stocking and dizziness, Brown patch, R. solani MAFF 511103, and large patch of grass. (R. solani MAFF 305245) also showed an inhibitory effect. On the other hand, bacteria and yeast were found to have no selective activity against plant pathogens because they had no growth inhibitory effect.

On the other hand, in the case of GB-017, the genus Bacillus had a high growth inhibitory effect against Pythium sp., Which is the cause of the stocking and dysentery, and of Botrytis cineria and wilted disease. Inhibitory effects were also shown for Fusarium sp, causal brown patches (R. solani MAFF 511103) and large patches (R. solani MAFF 305245). It was found to have selective activity only against plant pathogens because it had no inhibitory effect on bacteria and yeast.

Example 4

[Culturing and Formulation of Antagonist Microorganisms]

Bacillus subtilis GB-0365 and genus Bacillus GB-017 were incubated using a 500 L fermenter, in which a liquid medium was used. g was mixed with 1 L of distilled water, and then adjusted to pH 7.0. Species culture was a Erlenmeyer flask using yeast extract 0.5%, tryptone 1.0%, salt 0.5%. 1 liter of seed culture cultured at 30 ° C. and 200 rpm for 12 hours was inoculated into a 50-l fermenter containing 30-l production medium, pre-incubated for 12 hours at 120 rpm and 1vvm at 30 ° C., and then containing 400-l production medium. It was inoculated in a 500 L fermenter and cultured under conditions of 30 ° C., 120 rpm, and 0.5 vvm until the highest antagonistic activity (14-16 hours from the start of fermentation).

The culture was prepared as a solution or granule microbial preparation, depending on the application. The composition of the microbial preparation of the liquid formulation and granules is shown in Table 5 below.

Example 5

[Effectiveness of Grass Turf Control in Greenhouse]

Using the liquid microbial agent, the major grass pathogens (Helminthosporium dictyoides MAFF 511103), Brown patch (Rhizoctonia solani MAFF 305245), Large patch (Rhizoctonia solani MAFF 305219), and Pythium aphanidermatum 199 for the Pythium aphanidermatum The antagonism was tested by Disclosure strains were grown for 2 weeks at 25 ℃ using bran medium (100 g of wheat + 100 ml of water). The culture was air dried and filtered with 8 mesh to use as inoculum for subsequent experiments. The small pot was filled with 1/2 volume of the sterilized soil, and the other half was mixed at the rate of 0.1 g of inoculum per 50 g of sterilized soil to fill the artificially infected soil. Then, the healthy grass was obtained, the soil at the root region was removed, and planted in a prepared pot. The untreated group did not use liquid microbial preparation after the formulation, and the treated group was diluted with 1 × 10 ⁶ cfu / g soil and sprayed 5 times with the standard amount of the microbial agent, and the presence of development was observed. The control group was treated with only four types of grass pathogens and no microbial agents were sprayed, and the test group was sprayed with the microorganisms while the incidence was higher than 80% by four types of grass pathogens. The results of investigating the effect of inhibiting the onset of turfgrass disease using the microbial agent prepared in the pot test are shown in Table 6 below. In the control group, the disease showed 100% incidence after 3 ~ 4 days of treatment, whereas in the control group treated with antagonist microorganisms, the effect of inhibiting disease was apparent up to 30 days.

Example 6

[Control Effect during Storage after Citrus Harvest]

We investigated the possibility of biological control by treating liquid microorganisms for Hayward varieties with the highest quality and storage among the promising citrus varieties as economic fruit crops in Korea. The naturally occurring molds of citrus fruits were Pennicillium sp., Trichoderma sp., Fusarium sp., Alternaria, etc. It was made, and inoculated in the lesion formed in two places at intervals of 2cm on the equatorial plane. The experimental groups were treated with untreated, liquid microbial immersion treatment, fungal combined inoculation, fungal combined inoculation and liquid microbial immersion treatment. In the untreated plot, mold developed only after 4 days of storage, and 37.76% of the total infection occurred during 2 weeks of storage. In addition, the treatment group immersed in the liquid microbial agent was significantly slower in the development of mold than other treatments. Specifically, after 8 days, it started to appear slightly at the connection site, but did not show other infection symptoms during the storage period of 2 weeks. In addition, there was no bacterial toxicity in the treatment group immersed in the liquid microbial agent except for the symptoms caused by the infection of the fungus. The fungal inoculated group showed signs of infection in the lesions after 3 days of storage, and the infection spread to the entire stomach on day 7 of storage. On the other hand, in the treatment group treated with liquid microbial agent and inoculated with the fungus, the spread of infection was slower than the untreated or fungus treatment group, and infection symptoms began to appear on the 6th day of storage.

As described in detail above, using the microbial agent according to the present invention, the active ingredient produced in the culture solution contained in the microbial agent acts primarily to inhibit the growth of mold, and secondly, the microorganisms in the microbial agent are applied in the field of application. While inhabiting, the active ingredient is produced to inhibit the growth of fungi, thereby reducing the incidence of diseases of various crops.

Claims (3)

Microorganisms Bacillus subtilis for controlling fungi causing crop diseases GB-0365 KFCC-11071. Bacillus subtilis GB-0365 KFCC-11071 with 8 g of soy flour, 10 g of glucose, 5 g of ammonium sulfate, 3 g of calcium phosphate, and 1.5 g of magnesium sulfate in 1 liter of distilled water, and then in a liquid medium adjusted to pH 7.0, A culture obtained by culturing under conditions of 30 ° C., 120 rpm, and 0.5 vvv up to 14 to 16 hours from the start of fermentation. Bacillus subtilis GB-0365 KFCC-11071 or as a active ingredient for plants gray ash disease, green disease, wearing bottle, wilting disease, brown patch, large patch control and fresh vegetable after harvest disinfectant.