KR20000050142A - method for producing a microbe agricultural with porosity microelement - Google Patents

Abstract

PURPOSE: An antifungal microbial pesticide coated on ceramics containing a porous trace element and a method for the preparation thereof are provided for inhibiting the reproduction of pathogenic mold microorganism of a plant as well as for supporting the increase of bioactivity of antagonistic microorganisms and a promotion of plant growth. CONSTITUTION: A microbial pesticide comprises the concentrated antagonistic microorganisms, 5-10 % of saccharides and organic acids, 4-6 % of nucleic acids, 50-55 % of iron oxide, 20-25 % of magnesium oxide, 3-5 % of manganese oxide, 10-15 % of zinc oxide and 0.1-1 % of the porous trace element, wherein the microorganisms include Pseudomonas maltophilia AG-12(KFCC10809), and mutants thereof, P. maltophilia TM-23, TM-24 and TM-25. The preparation method comprises: concentrating a microorganism culture; adding the saccharides and organic acids to the culture; mixing the culture with carrier; lyophilizing the mixed culture and then obtaining the microorganisms; stabilizing the microorganism by mixing the microorganisms with ceramics components.

Description

Antifungal microbial pesticides coated on ceramics containing porous microelements and a method for producing the same {method for producing a microbe agricultural with porosity microelement}

The present invention relates to an immobilized microbial pesticide immobilized on a carrier, and more particularly, to an antifungal microbial pesticide coated on ceramics containing a porous microelement by immobilizing an antagonist microorganism on a porous far-infrared ceramic carrier and a method of manufacturing the same.

Until now, microbial pesticides have been exposed to problems such as incomplete delivery system and poor effectiveness as pesticides. Therefore, attempts to improve the microbial pesticide delivery system have been continued in recent years. As an example, the use of porous carriers based on peat, carrageenan and alginic acid has already been reported. However, most of these methods have improved formulation properties, and such processing involves a difficult process, resulting in high manufacturing costs, and unsatisfactory delivery systems.

In vegetable crop farming in Korea, there are damages caused by red pepper germ disease, cucumber manger germ, and Chinese cabbage germ, but until now, chemical pesticides have been mainly used to control these germs. However, the continued use of pesticides has caused various problems due to soil and river contamination and pesticide residues, which raises the interest in controlling plant diseases using microbial agents, and the demand for them is increasing day by day.

Recently, biological control of plant diseases using antagonistic microorganisms has been heightened as an alternative stage to solve various problems caused by overuse and contamination of organic synthetic pesticides, and it is used for biological control by directly or formulating antagonistic microorganisms separated from soil. Or mutants are being promoted to enhance antagonism. In addition, research on low-toxic or pollution-free microbial pesticides is being actively conducted.

Carriers, nutrients, and protective agents, which are generally used as adsorbents, are used for the preparation of microbial pesticides. In general, inorganic and organic materials are used as carriers. Bumcurite, zeolite, diatomaceous earth, Calcium carbonate and rice bran, bone meal, chitan and peat are mainly used, and many other materials are used.

In addition, as a material required for formulation, stabilizers necessary for stabilization of microorganisms, and trace elements to help adsorption and supply of microorganisms are used.

Conventional formulation methods have been widely used in the formulation method of mixing the microbial culture medium with the carrier and stabilizer, nutrients, trace elements and the like, then dried to powder or liquefied in a liquid state.

However, the conventional formulation method has a lot of problems in microbial formulation because the microorganisms do not sufficiently reach the low temperature, dehydration and the resting state of the cells, some of them are killed or disturbed depending on the type.

Under these circumstances, the present inventors have shown that antagonism microorganisms can be used as a more practical and simpler method than conventionally known microbial agents for the purpose of promoting plant growth and controlling various diseases, and also effectively controlling plant diseases without damaging environmental pollution. The present invention has been completed as a result of intensive studies to provide a microbial formulation stabilization method and a microbial formulation using.

Therefore, the present inventors have studied to develop a practical microbial pesticide delivery system, and as a result, when the antagonist microorganisms are fixed to the porous ceramic containing trace elements, the present invention is transferred to plant pathogens and molds present on the soil or plant surface around the crops to provide sufficient sterilization effect. Particularly, when the ceramic used as a carrier is composed of natural products, it is found that it is very economical as a material that is pollution-free and relatively low in manufacturing cost, thereby completing the present invention.

That is, it is an object of the present invention to provide an immobilized microbial pesticide made by immobilizing antagonist microorganisms in a porous and trace element-rich ceramic or a workpiece thereof.

It is another object of the present invention to provide a method for producing an immobilized microbial pesticide made by immobilizing an antagonist microorganism in a porous and trace element-rich ceramic or a workpiece thereof.

1 is a graph of changes in tomato height according to an embodiment of the present invention

Figure 2 is a graph of changes in tomato leaves according to an embodiment of the present invention

3 is a diameter diagram of the tomato according to an embodiment of the present invention

4 is a graph of the resultant leaves of radish according to an embodiment of the present invention

5 is a graph of rhizome results of radish according to an embodiment of the present invention

6 is a graph of the result of growing cucumber according to an embodiment of the present invention

7 is a manufacturing process flow chart of an embodiment of the present invention.

The present invention is proposed in the Republic of Korea Patent No. 130075, Pseudomonas maltophilia AG-12 strain (KFCC-10809) having a control activity in plant diseases and mutant strains to increase the control activity by introducing transposon Tn5 to the strain It is manufactured by using TM-23, TM-24, TM-25, and sprays on the plant cultivation of plants that are concerned about or have plant diseases to effectively control plant diseases.

The present invention for achieving the above object is a microelement specially processed Pseudomonas maltophilia AG-12 strain (KFCC10809) and mutant strains TM-23, TM-24, TM-25 having a control against plant diseases It is a pollution-free and economical immobilized microbial pesticide immobilized on a carrier made of a porous ceramic containing.

This invention makes it possible to provide a microbial formulation which complements the problems with the stabilization of microorganisms seen in prior formulation techniques and allows for longer viability to be preserved.

The mutant strain TM-23, TM-24, TM-25 shows a wide range of strong antimicrobial activity against red pepper germ disease, cucumber man germ, Chinese cabbage disease, which causes great damage to horticultural crops, mutant organic processes become parent Pseudomonas maltophilia AG-12 strain was suspended in 10 ml of LB liquid medium (10 g of tryptone, 5 g of yeast extract, 10 g of NaC1) to a concentration of 5 x 10 ⁸ cells / ml, and the concentrated P1 phage vector SF 800 was 25 µg. Incubate at 31 ° C. for 30 minutes, add 1 ml LB liquid medium, and incubate for another 2 hours at about 30 ° C., and 10 µl of the culture solution was obtained using X-gal and IPTG (isopropyl β-D-thiogal). It is selected to form blue colonies by plating on LB plate medium (tryptone 10g, yeast extract 5g, NaC1 10g, bacto agar 15g) to which lactopyranoside) is added and incubating at about 37 ° C for 2 days.

In order to confirm the insertion site of Tn5 lac, the antagonistic-strengthened mutant strains isolated the chromosomal DNA of each mutant strain TM-23, TM-24 and TM-25, respectively, were digested with EcoRL restriction enzyme, and 0.8% agarose gel. After electrophoresis, the DNA on the agarose gel was denatured with 0.5 N NaOH, 1.5 M NaCl, neutralized with 1.5 M NaCl, 0.5 M Tris-Cl (pH 7.5) solution, and then adsorbed onto a Nitran membrane. I was. The Tn5 lac insertion site was identified by Southern hybridization method using pRZ 102 (ColE1: Tn5) labeled with 32P isotope as a probe using DNA-adsorbed nitrile membrane, and Tn5 lac in all three strains. It was confirmed that it was inserted.

The mutant strains were mentioned in Korean Invention Patent No. 130075, and the contents of the isolation and identification of antagonists were also reported in detail.

In the preparation method of the present invention, the microbial culture solution is first concentrated in order to overcome the problem that the microorganisms are not sufficiently reached the low temperature, dehydration, and the resting state of the cells in the conventional formulation method described above. Sugars dextrose, sucrose, lactose, meso-inositol, etc. are added to the concentration of microorganisms at a concentration of 5-10%, amino acid glutamic acid is added at a concentration of 5%, mixed with a carrier such as jeosil, and frozen. The microorganisms are first prepared by the drying method, and then the carriers of the ceramic component are mixed in an appropriate amount to stabilize the microorganisms.

Hereinafter, the present invention will be described in detail.

The method for preparing a microbial pesticide preparation of the present invention comprises the steps of concentrating a microbial culture solution in the preparation of a microbial pesticide by immobilizing the antagonist microorganisms (S1); Mixing saccharides, organic acids and nucleic acids in a microbial culture solution concentrated in the step (S1) (S2); Mixing the microbial culture solution obtained by the step (S2) with a carrier (S3); Obtaining a microorganism by freezing and drying the microbial culture solution mixed with the carrier by the step (S3) (S4); It is prepared by the step (S5) of stabilizing the microorganism by mixing a ceramic component with the microorganism obtained by the process (S4).

Such a microbial pesticide preparation of the present invention in the preparation of a microbial pesticide by fixing the antagonist microorganisms in the adsorbent, by mixing 5-10% of saccharides and organic acids, 4-6% nucleic acid in the concentrated antagonist microorganisms fixed in the adsorbent and frozen And dried, and 50 to 55% of iron oxide (Fe ₂ SO ₃ ), 20 to 25% of magnesium oxide (MgO ₂ ), 3 to 5% of manganese oxide (MnO ₂ ), 10 to 15% of zinc oxide (ZnO), It is characterized in that the immobilized on the ceramic particles consisting of 0.1 ~ 1% of the porous trace elements of the inorganic salts.

Generally known carriers are mainly used natural products such as cereals, tubers, tubers, and many other by-products such as rice bran, wheat bran, barley bran, straw, barley straw, rice straw and the like. In particular, as a microbial stabilizer, glycerol, skim milk, etc., which are excellent for stabilization, are used, but there is a problem in that the economy is poor.

The present invention used a ceramic particle different from the above carriers as a carrier for immobilizing the antagonist microorganisms, the composition of which is 50 to 55% of iron oxide (Fe ₂ SO ₃ ), 20 to 25% of magnesium oxide (MgO ₂ ), Manganese oxide (MnO ₂ ) 3 to 5%, zinc oxide (ZnO) 10 to 15%, and is composed of 0.1 to 1% of TiO ₂ , Al ₂ O ₃ , SiO ₂ , ZrO ₂ , CaO as trace elements. In addition, it is a material that helps stabilize the antagonist microorganisms is completed by mixing 5-10% of sugars and organic acids, and 4-6% of nucleic acid.

As described above, the microbial pesticide preparation of the present invention comprising the ceramic carrier exhibits the following effects.

Far-infrared radiation ceramic used in the present invention emits far-infrared rays between 5.6-20 μm, and this wavelength range has various physical effects. In the crop test results using radioceramic, the number of days of hair growth as a source of energy was shortened, and it has been reported that there is much effect on seed germination and seedling growth by the heat action. In particular, when directly projected onto plant cells, they cause resonance and resonance, which greatly affects the activation of living cells. In addition, the release of radiant energy of the ceramic particles increases the ability of fixing carbon dioxide on the leaves, so that photosynthetic activity occurs actively, so that the content of carbohydrates (glucose, starch) in the plant is increased, and leaf and stem growth are vigorously generated. In particular, the thicker the leaves, the thicker the stem, and helps the development of the muscles.

As described above, the immobilized microbial pesticide of the present invention is a microelement in the carrier at the time when the antagonistic microorganisms grow to act as a nutrient by the metabolism of the microorganisms, as well as the growth of microorganisms and the production of bioactive substances In addition, the secretion of bioactive substances from the microorganisms and the spread of the microorganisms are slowed, so that the effects of the pesticides can be sustained for a long time. Moreover, both microorganisms and carriers are natural products, so there are no environmental pollution or pollution problems, and they are very economical using cheap carriers.

Hereinafter, the specific examples of the present invention will be described through manufacturing steps and test examples. However, the present invention is not limited to the examples, and in particular, the antagonists and carriers used may be variously selected and used according to the purpose of the present invention.

In the following examples, the sugars may be selected from all kinds of sugars including glucose, sugar, taxrin, and molasses, and the organic acid is arginic acid and the nucleic acid is glutamic acid.

[Step 1]

Fill a 1 L Erlenmeyer flask with 200 ml of sterile LB medium (tryptone 10 g, yeast extract 5 g, NaC1 10 g, pH7.0), followed by Pseudomonas maltopila AG-12, KFCC10809 (0.4 at 600 nm) 20 Inoculated with ml and incubated for about 24 hours at 30 ℃. The cultured cell solution was 7000 rpm in a centrifuge to separate the cells and the culture medium for 20 minutes, and recovered and used only the separated cells. In stabilizing the recovered cells, 5% to 20% of glycerol was added to the homogenized state. Antagonist microorganisms were prepared according to the experimental purpose. In addition, depending on the purpose of use it was used by air drying or freeze drying or high temperature drying.

[Step 2]

Raw materials → calcining → quantitative mixing → 1st milling → stirring and maturing → 2nd milling → stirring and maturing → drying → 3rd milling → 4th milling → inspection, etc. Fe2SO3) 50-55%, magnesium oxide (MgO) 20-25%, manganese oxide (MnO2) 3-5%, zinc oxide (ZnO) 10-15%, TiO2, Al2O3, SiO2, ZrO2, CaO are mixed properly The amount of the trace element is 0.1-1%, and in particular, these particles are pulverized after heating at 800 ° C to 1250 ° C and aged for 3 to 5 days to prepare a porous ceramic having a desired particle size.

[Step 3]

In blending the antagonists and porous ceramics prepared in the above steps 1 and 2, the most necessary combinations of plants and antagonists are quantitatively mixing in the ratio of each composition to antagonist microorganisms at a temperature of 30 ℃ 24 Aged and dried over time to obtain an immobilized microbial preparation. The concentration of microorganisms thus obtained is 108-109 cells per gram, and the effective concentration is 106-107 cells per gram.

[Test Example 1]

As a microbial agent obtained in step 3, the effect of control activities on phytopathogens, red pepper bacillus (Phytophthora capsici), cucumber manganese (Fusarium oxysporum), and cabbage (Rhizoctonis solani) were performed. The effective concentration was used by diluting the microbial agent to 106 ~ 107 cells per gram and compared the test by the size of growth inhibitory observed for each fungus.

[Test Example 2]

Each mold culture medium is drilled about 1 cm in diameter, and the bottom of the hole is treated with a small amount of 0.8% Agar so that there is no gap. 200 μl of Pseudomonas maltophilia cultured at 28 ° C. for 24 hours was treated with LB medium (Bacto-tryptone 10g, Bacto-Yast extract 5g, NaCl 10g). After drying it was completely plugged with 0.8% Agar.

In addition, the fungus spores grown on a solid medium were mixed with 0.8% Agar and poured thinly on a test solid medium prepared in advance, and then hardened and incubated at a growth temperature of 28 ° C. for 48 hours to observe growth inhibition rings.

The growth inhibitory ring size observed for each fungus is shown in Table 1.

The size of the growth ring of each fungus. Pathogenic fungus Size of growth refrain ring Phytophthora capsici 23 Fusarium oxysporum 24 Rhizoctonia solani 26 Botrytis cinerea 24

[Test Example 3]

Hydroponic cultivation of crops such as tomatoes, radishes, cucumbers among fruits and vegetables, and the microbial agent prepared in step 3 to the nutrient solution and leaves of the crops were attached to the graph to investigate the effect on the growth and fruit of the crops.

Figure 1 shows the change in tomato height (crop height) showed a change in crop height.

Figure 2 shows the change in the tomato leaves, but there was no significant change in the leaves, but the increase in chlorophyll and the leaves became noticeable.

Figure 3 shows the tomato fertilization was confirmed that the leaves thickened and chlorophyll increased.

Tomato growth results are shown in Table 2 below.

The growth of tomatoes showed effects on leaves, stems and roots. In other words, the leaves become lush.

4 shows that chlorophyll increases and leaves become thicker as a result of the leaves of radish.

Fig. 5 shows a difference of about 10% as a result of rootless rooting.

In the following Table 3, the fresh weight (g) of radish is shown.

The freshness of radish was especially effective in the second treatment, and it was effective in both roots and leaves.

6 shows the results for the growth of cucumbers.

The present invention promotes the growth of plants, has a control effect on various diseases, and stabilizes and formulates microorganisms using antagonistic microorganisms that do not cause environmental pollution, while mixing sugars and nucleic acids with primary concentrated microorganisms and mixing them with carriers. By freezing, the microorganisms do not reach the resting state of the cell and die by dehydration, thereby achieving stabilization of the microorganisms. Also, by fixing to a porous ceramic, a natural substance containing a trace element as a carrier, it is pollution-free and relatively inexpensive to manufacture. As a material, it can exhibit sufficient bactericidal effects against plant pathogens present on the soil or plant surface around the crop.

Claims (2)

In preparing microbial pesticides prepared by immobilizing antagonist microorganisms in adsorbents, 5-10% of saccharides and organic acids and 4-6% of nucleic acid were added to the concentrated antagonist microorganisms, fixed in an adsorbent, and freeze-dried, and 50-55% of iron oxide (Fe ₂ SO ₃ ) and magnesium oxide (MgO ₂ ) were added thereto. Microporous traces characterized by immobilization on ceramic particles consisting of inorganic salts of 20 to 25%, manganese oxide (MnO ₂ ) 3 to 5%, zinc oxide (ZnO) 10 to 15% and porous trace elements 0.1 to 1% Antifungal microbial pesticides coated on ceramics containing elements. In preparing microbial pesticides prepared by immobilizing antagonist microorganisms in adsorbents, Concentrating the microbial culture; Mixing saccharides, organic acids and nucleic acids in the microbial culture medium concentrated in the step; Mixing the microbial culture obtained by the step with a carrier; Obtaining a microorganism by freezing and drying the microbial culture solution mixed with the carrier by the above steps; Method for producing an antifungal microbial pesticide coated on a ceramic containing a porous microelement, characterized in that the microorganisms are stabilized by mixing a ceramic component with the microorganism.