KR100991298B1 - Biotic pesticide using Arthrobacter zoysiae with inhibition activity of germinaton and growth of seeds - Google Patents

Abstract

The present invention relates to a biological pesticide capable of inhibiting germination and growth of seeds containing the new strains Arthrobacter zoysiae YC 6002 and Arthrobacter zoysiae or its culture solution as an active ingredient.

According to the present invention, seed germination and growth of weeds can be suppressed in crop cultivation using Arthrobacter zoysiae, the new strain of the present invention excellent in germination and growth inhibition of seeds, and weeds in crop cultivation. Reduced damage can increase the production of agricultural products.

Atsobabacter sieshi, seed, germination, growth inhibition, biological pesticide, microbial herbicide

Description

Biological pesticide using Arthrobacter zoysiae with inhibition activity of germinaton and growth of seeds}

The present invention relates to a biological pesticide capable of inhibiting germination and growth of seeds using the new strain Athrobacter zoey, and more specifically, to a seed containing an Asthrobacter zoysiae strain or a culture thereof as an active ingredient. It relates to a biological pesticide with germination and growth inhibition.

Korea has used a large amount of chemical pesticides to control diseases, pests and weeds every year to increase the yield of crops such as cereals, vegetables, and fruit trees for the past several decades. Issues such as destruction, poisoning toxicity, causing pests and weeds to resist pesticides continue to be raised.

The government is enacting the Eco-Friendly Agriculture Promotion Act in 1999 to reduce the use of about 1 trillion chemical pesticides and improve the competitiveness of our agriculture. Pesticide use is not decreasing.

However, the demand for safe food is increasing due to the improvement of people's living standards, and eco-friendly agriculture such as organic farming and natural farming, which are aimed at maintaining the agricultural production environment and preserving the natural ecosystem, is rapidly expanding, replacing chemical pesticides. Research into the development of biopesticides that can be done is increasing rapidly.

Biopesticides control pests by mass-cultivating and formulating natural insects and antagonistic microorganisms and treating them with foliar spray or granulation, and weed pathogens may be used for weed control. The microorganisms used here are widely distributed in the natural world, and various kinds thereof are used to secrete metabolites that exhibit pest and weed suppression effects, and thus many kinds are used for biopesticide development.

There are three ways to use such microorganisms: microorganisms themselves, metabolites produced by microbial fermentation, and microbial metabolites as the leading compounds for new pesticide synthesis. Among them, many studies have been made at home and abroad on phytopathogenic microorganisms capable of inhibiting phytopathogens, plant growth promoting myobacterial bacteria (PGPR) promoting plant growth, and plant endogenous bacteria.

Plant endogenous bacteria are defined as bacteria that live in living healthy plant tissues and do not cause substantial harm to plants, but which have various benefits. Generally, plant endogenous bacteria are mostly collected in the spaces between plant cells, but some types of cells Some endogenous bacteria are known to have direct or indirect resistance to environmental stresses of plants, and are known to have a great effect on plant growth.

Plant endogenous bacteria are present in the cell and proliferate. In general, the total density is highest at the beginning of the roots and stems, and gradually decreases toward the top of plants such as stems and leaves, and in the case of corn leaves, the density is Log 3-7 CFU / g. It is enough. To date, more than 129 species of endogenous bacteria have been known, including Pseudomonas, Enterobacter, Bacillus, and Agrobacterium. These include tomatoes, lettuce, potatoes, and corn. Increasing the production of crops by inhibiting the growth and disease outbreak of economically important crops such as rice and rice.

However, there are relatively few studies on microorganisms that inhibit plant growth or microbial herbicides using the same. In particular, research on microbial herbicides has been conducted by several researchers, but it has not been developed as a product yet. In other countries, products such as Colego and Devine have been developed and sold. Registration exams in each country. All of these microbial herbicides use phytopathogens that cause diseases in the target weeds, and no herbicides using plant endogenous bacteria have yet been developed.

On the other hand, the greatest advantage of the microbial herbicide is that it does not cause environmental problems when used, and in most cases it is specific to only the weeds of interest to have high selectivity. However, microbial herbicides have difficulty in development because they have a narrower control spectrum and late effects than chemical pesticides.

In contrast, the endogenous bacteria of the present invention can be developed as a non-selective herbicide because it suppresses both the growth of the target weeds and crops.

Accordingly, the present inventors have made diligent research efforts to overcome the problems of the prior arts, and as a way to compensate for this, Asthrobacter Joysi can broadly suppress seed germination and growth of various plants including weeds. By separating and mass cultivating zoysiae), it was confirmed that a new type of microbial herbicide could be developed and the present invention was completed.

Therefore, the main object of the present invention is to provide a biological pesticide using Athrobacter Joysy which can suppress the seed germination and growth of various plants including weeds.

It is another object of the present invention to suppress seed germination and growth of weeds in a crop plantation using biopesticides using the Asrobacter zoey, and to increase the production of agricultural products by reducing the damage caused by weeds during crop cultivation.

According to an aspect of the present invention, the present invention provides a biopesticide for inhibiting germination and growth of seeds, characterized in that it contains an Asthrobacter zoysiae strain or a culture thereof as an active ingredient. The strain of the present invention can be used as a non-selective herbicide because it suppresses both the growth of the weeds and crops of interest.

In the biopesticide of the present invention, the Asthrobacter zoysiae strain has the morphological and physiological characteristics shown in Table 1 below.

Table 1 Morphological and Physiological Characteristics of Asthrobacter zoysiae YC 6002 Strains

In the biological pesticide of the present invention, the Arthrobacter zoysiae YC 6002 includes a parent strain and a mutant strain, but preferably has a 16sRNA of SEQ ID NO: 1, and Korea Biotechnology Research Institute Bioresource Center ( The strain deposited with the accession number KCTC 18127P in KCTC: Korean Collection for Type Cultures.

In the biological pesticide of the present invention, the Asthrobacter zoysiae strain or its culture may be variously modified by a known method used in the art as a liquid and powdered form, but is preferably used. It is preferable to adsorb and dry the culture or concentrate of the Asthrobacter zoysiae strain on a carrier such as starch, crude protein or rock meal. The carrier which can be used in admixture with the culture or concentrate of the strains of the invention can be any carrier used in the art. Specifically, those that can be used as a carrier in the present invention include rice, wheat, corn, barley, soybean, crude, sorghum, millet, grains such as buckwheat, tubers such as potatoes, tubers such as sweet potatoes, cassava, or processed products thereof. (Eg, powder), starches derived from them, derivatives thereof, and the like. In addition, diatomaceous earth materials such as agar, gelatin, pactate (polygalacturonate), chitosan, carboxymethyl cellulose and its derivatives, gelatin, natural wax, natural gum, kaolin, bentonite and other clay minerals, or geolites, may be used. Can be. These various carriers may be used individually, or two or more carriers may be mixed in an appropriate ratio to obtain an improved physical carrier. If such carriers are used, they may be metabolized by microorganisms to become nutrients, and these carriers may be highly viscous to increase adhesion to the surface of the plant.

According to another aspect of the present invention, the present invention provides Arthrobacter zoysiae YC 6002 deposited with the Korean Collection for Type Cultures (KCTC) Korean Deposit for Type Cultures (KCTC 18127P).

Hereinafter, the configuration of the present invention will be described in more detail.

In the present invention, it was intended to isolate and identify endogenous bacteria that inhibit germination or growth of plant seeds and to develop microbial herbicides through mass culture and formulation of these bacteria. In order to develop a new type of microbial herbicide, endogenous bacteria of various plants that inhibited seed germination and growth were isolated from 20 plant tissues near Jinju Jinju. The seed germination inhibitor of the plant was examined using 300 strains among the various isolates, and among them, the strain of Arthrobacter zoysiae of the present invention having the best germination inhibitor was selected from the root tissue of grass. It was. In order to know the effect of inhibiting seed germination using the Arthrobacter zoysiae YC6002 strain culture solution of the present invention, the inhibition of radish seed, a dicotyledonous plant, was investigated. As a result, the strain of Asthrobacter zoysiae (Arthrobacter zoysiae) YC6002 was examined. The effect of seed germination inhibition was confirmed. In addition, in order to know the main monocotyledonous and dicotyledonous weeds and crop seed germination inhibitory effect of the strain of the present invention, the culture medium of the strains is the monocotyledonous weeds (sorghum, blood, wheat, barley, American dog field) and dicotyledon weeds The germination inhibition of all seeds was confirmed before the germination of rice, corn and cucumber) and rice, corn and cucumber.

Seed germination inhibitory effect of the strain of Arthrobacter zoysiae YC6002 of the present invention according to the culture temperature was all inhibitory effect for 72 hours incubation at 20 ℃ ~ 30 ℃ culture temperature, 1 day as a result of examining the germination inhibition by period Afterwards, the germination inhibitory effect was higher than that of the control.

Physiological and biochemical characteristics of the isolated endogenous bacteria YC6002 strain of the present invention were grown to 15-37 ℃ as aerobic anaerobes, the form was Gram-positive. The sequencing of 16S rDNA showed 96-98% similarity to the standard strain of Arthrobacter sp. The preliminary analysis showed that the physiological and biochemical and molecular biological characteristics were similar to those of YC6002. Analysis of fatty acids, phospholipids, and DNA-DNA hybridization using an Asthrobacter strain showed that YC6002 was finally identified as a new species of the genus Arthrobacter sp. (Arthrobacter zoysiae) YC6002.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1 Selection and Identification of Seed Germination Inhibiting Bacteria YC6002 of the Present Invention

1. Selection of Germ-inhibiting Germ-free Seeds

In order to isolate endogenous bacteria that inhibit seed germination, 20 plants including soybean, corn, cabbage, and grass were collected from pearls and various parts of Sichuan. To isolate symbiotic endogenous bacteria in plant tissue, cut the root of the plant by 1g and wash it in running water, soak it in 1% sodium hypochlorite (NaOCl) solution for 1 minute, and then in 70% ethyl alcohol solution. After a few seconds of surface sterilization and rinsing in sterile distilled water.

Add 1 ml of sterile distilled water to 1 g of the plant root prepared as described above, grind it into a mortar, take 1 ml of this, and dilute it to 105 times, then 0.1 trytic soy broth 3 g, agar 18 g / sterile distilled water 1 L. 0.1 ml) was spread evenly over the whole surface of the medium and then cultured in a 30 ° C. incubator for 3 days to isolate a single flora of growing bacteria.

The isolated strains were grown again in 0.1TSA medium to confirm that the bacteria were pure, and then cultured in a slope medium and stored at 4 ° C. and used for the experiment.

By the above method, approximately 300 strains among various colonies of different floras and colors are purely separated and cultured in 0.1 Triptic Soy Broth (TSB, 3g / sterilized distilled water) to test seed germination inhibition. Among them, YC5957, YC5965 and YC6002 strains were good germination inhibitory effect. In particular, the YC6002 strain did not germinate at all after 5 days.

In addition, the strains were cultured by replacing the strains with phytopathogenic fungi Collectorcumum coleosporioides in 0.1TSA and PDA medium, and the degree of pathogen inhibition was also investigated. The strains of five strains including YC5957 and YC5975 strains were also inhibited. The effect was also excellent (Table 2).

[Table 2] Inhibition of Germination of Mycelial Growth and Radish Seeds by Isolated Endogenous Bacteria, Colletotricum Gloeosporioides

2. Identification of Germ-inhibiting Germ-free Seeds

(1) Physiological and biochemical characteristics of seed germination inhibitory endogenous bacteria YC6002 strain

First, the morphology of seed germination inhibitory endogenous bacteria was attempted by optical and electron microscopy. After staining the strain by Gram staining method, Gram positive or negative was observed under an optical microscope, and electron microscopic observation showed that the strain was fixed with 2.5% glutaraldehyde and osmium tetraoxide. Observation was made using an electron microscope (Philips, XL30 S FEG). The pretreatment of the samples was done by routine scanning microscope sample preparation of the joint laboratory of Gyeongsang National University.

As a result of microscopic examination, the YC6002 strain was aerobic in the form of Gram-positive bacillus, and it could be grown from 25 ° C to 35 ° C, and the optimal growth temperature was 30 ° C. Catalase was positive and oxidase was oxidase. It was negative (Table 1, FIG. 1).

Next, the biochemical characteristics of the seed germination inhibitory endogenous bacteria were investigated. Physiological examination of strains is described by Smith, RM and Krieg, NR 1991. General characterization, p. 409-433 .; Murray et al (ed.), Manual of methods for general bacteriology, American Society for Microbiology , Washington, DC) were subjected to oxidase, catalase, urease reaction, casein, starch digestion test, and the biochemical properties of the prokaryotic system [Chemical] methods in prokaryotic systematics (Goodfellow, M. and O'Donell, AG 1994. Chemical methods in prokaryotic systems. John Wiley & Sons, New York, NY, p. 575). Important physiological and biochemical properties were investigated using the API (Analytic Profile Index) ID 32E kit (bioMerieux, Marcy-I'Etoile, France).

Various physiological and biochemical characteristics of the ID32E kit and the results of the examination were as shown in Table 1 and Table 3 below.

Table 1 Morphological and Physiological Characteristics of YC6002 Strains

+; Positive reaction,-; Negative response

[Table 3] Carbohydrate utilization reaction by endogenous bacteria YC6002

* Confirmation after 24 hours incubation at 37 ° C. using API ID 32E identification system.

+; Positive reaction,-; Negative response.

(2) Molecular Biological Characteristics of Endogenous Bacteria YC6002 Strains

One) Endogenous bacteria YC6002  16S of strain rDNA  Sequencing

Primers for 16S rDNA amplification and sequencing of isolated strains were used as the universal primer set 27F / 1492R (5`-AGAGTTTGATCMTGGCTCAAG-3` / 5`-GGYTACCTTGTTACGACTT-3`) ) Was used. Each primer was diluted to 100 μM and stored at −20 ° C. and used for the experiment. PCR was amplified using the primers above. Denaturation, annealing, and extension were 94 ° C. for 1 minute, 56 ° C. for 1 minute, and 72 ° C. for 1 minute. After repeated 30 times and reacted for 10 minutes at 72 ℃ to amplify the 16S rDNA gene. The obtained base fragments were determined by an automatic sequencer, and the database search was performed using the BLAST program of NCBI network service and RDP analysis tool of Ribosomal Database Project II.

The nucleotide sequence of the 16S rDNA gene of the endogenous bacterial YC6002 strain (SEQ ID NO: 1) showed 96-98% similarity to the standard strain of the genus Arthrobacter sp.

In addition, the 16S rDNA sequence of the YC6002 16S rDNA sequence of the isolated bacterium was sequenced by using a gene bank database (Genebank Data base) multiple strains similar to these sequences to obtain similarity (similarity). Phylogenetic tree was prepared using the base sequence, and the result of generating the phylogenetic tree using PHYLIP pakage for analysis is shown in FIG. 2B. In addition, it can be seen that the strain YC6002 is similar to the strain Athrobacter globiformis, which is a standard strain of the genus Arthrobacter sp.

2) Further analysis of biochemical properties of seed germination inhibitor YC6002

Among the plant endogenous bacteria isolated, YC6002 was the best seed suppression effect, so further analysis was performed for accurate identification of YC6002. In comparison with the isolated YC6002 strain, one strain, which was the highest in 16S rDNA similarity in preliminary experiments, was distributed from Japan Collection of Microorganisms (JCM), Korean Collection for Type Cultures (KCTC) and Korean Agricultural Culture Collection (KACC). (Table 4). To confirm the identification of YC6002, fatty acid, phospholipid analysis, and DNA-DNA hybridization were performed using an Arthrobacter strain similar in physiological, biochemical, and molecular biological characteristics to YC6002.

 Table 4 Standard Strains of Arthrobacter Species Used in the Study

* JCM (Japan Collection of Microorganisms, Saitama, Japan), KCTC (Korean Collection for Type Cultures, Daejeon, Korea), KACC (Korean Agricultural Culture Collection, Suwon, Korea)

2) -a. Fatty Acid Analysis of YC6002 and Similar Strains

Fatty acids were extracted using the Microbial Identification System (MIDI) method for fatty acid analysis of YC6002 and comparative strains. Sample pretreatment was carried out in the order of harvesting, saponification, methylation, extraction and washing. 40 mg of cells incubated in TSA solid medium at 30 ° C. for 24 h were placed in a screw cap reaction tube. Saponification solution [NaOH 15 g, methanol 50 mL, D.W. 50 ml] was added and the lid was tightly closed and reacted at 100 ° C for 5 minutes. After 5 minutes, shake well for 5-10 seconds and react again at 100 ° C for 25 minutes. Thereafter, the temperature of the test tube was lowered to flowing water, and then, 2 ml of methylation solution [130 mL of 6N HCl, 110 mL of methanol] was added and sealed, followed by reaction at 80 ° C. for 10 minutes. After immersing in iced water, it was cooled quickly and the extraction solvent [hexane 100 ㎖, methyl-tert-butylether 100 ㎖] was added 1.25 ㎖ and shaken for 10 minutes. When the film was formed, the lower layer was removed with a Pasteur pipette, and about 3 ml of saturated NaCl was added to the remaining solvent layer, followed by stirring for 5 minutes. After that, only 2/3 of the solvent layer was removed and placed in a GC vial to prepare a sample. It was. The analysis of the samples was carried out using a microbial identification system (Sherlock, USA) of the joint laboratory of Gyeongsang National University.

Fatty acid analysis results of the five different strains from YC6002 are shown in Table 5. The most common fatty acids of YC6002 were 36.54% in 15: 0ai, 8.77% in 18: 0, 6.44% in 16: 0, and 5.07% in both 16: 1i and 16: 0i. The remaining 5 strains had the highest content of 15: 0ai as in YC6002, but the second highest content of A. globiformis was 21% in 15: 0i, 34.44% in A. koreensis and 34.44% in A. woluwensis. 15: 0i was 22%.

Table 5 Cellular Fatty Acids of Asthrobacter Species Similar to Plant Endogenous Bacteria YC6002

* Values lower than 1% are not shown.

2) -b. Comparative analysis of biochemical properties

Table 6 shows the results of analysis of biochemical properties of YC6002 and five other strains. YC6002 showed a bright yellow mycelial morphology, similar to A. koreensis with negative oxidase, trypsin and β-glucosidase. The G + C content was 61.2 (mol%), similar to the other 5 strains, and showed a very high G + C content. Overall, YC6002 did not differ significantly from the remaining 5 strains.

Table 6 Comparison of phenotypes between similar species of Arthrobacter sp.

* Expressed traits were compared using API ZYM, API ID 32E identification system.

+; Positive reaction,-; Negative response, c; cream-colored, y; yellow, EMB; eosin methylene blue,

One; YC6002, 2; A. globiformis, 3; A. koreensis, 4; A. pascens, 5; A. ramosus, 6; A. woluwensis

2) -c. Phospholipid Analysis by Two-dimensional TLC

Microorganisms in which phospholipids are most effectively utilized as biochemical taxonomic indicators of bacteria are Gram-positive bacteria. There are many methods for phospholipid extraction and analysis, but due to its simplicity, phospholipid analysis was performed by extraction using solvent fractionation and 2-Dimensional Thin Layer Chromatography (2D TLC).

Phospholipid extraction of cells was performed in the following manner. First, 500 mg of each cell cultured with shaking at 30 ° C. for 24 h in TSB medium was added with 2 ml of 0.3% NaCl solution, stirred well, and placed in a test tube for screw cap reaction. After adding 6.5 ml of methanol thereto, the cap was tightly closed and reacted at 100 ° C. for 5 minutes. After lowering the temperature of the test tube in flowing water, 3 ml of chloroform and 0.3% NaCl were added and shaken well for 2 to 3 hours in a shaker incubator. Add 3 ml of chloroform and 0.3% NaCl to each, and shake well. When the membrane is formed, only 2/3 of the lower layer is lifted up and concentrated with a rotary vacuum evaporator system (Eyela, Japan) to give chloroform-methanol (2: 1, v / v) was dissolved in 100 μl of the solvent and used for phospholipid analysis.

 Phospholipid analysis of the cells was performed by dropping 5 μl of the extracted phospholipid onto the edge of the horizontal × vertical 10 cm silica gel TLC plate (Silica gel 60F254, Merck, Germany) (1 cm below and to the left). chloroform-acetic acid-methanol-water (80: 13: 12: 1.6, by vol.) by one-dimensional development with water (65: 25: 3.8, by vol.) The two-dimensional development is done. After drying in the wind, evenly sprayed with a coloring reagent (phosphomolybdic acid, Sigma, USA) and heated at 150 ℃ for more than 15 minutes to analyze the phospholipids.

The phospholipid analysis revealed that the main phospholipids of YC6002 were phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidyl-N-methylethanolamine (PME) and ninhydrinphosphatidylglycerol (NPG).

2) -d. YC6002 and Arthrobacter sp. DNA-DNA hybridization of strains

Genomic DNA-DNA hybridization was performed by modifying the fluorescence microplate method of Ezaki et al. The genomic DNA of the extracted strain was diluted with phosphate buffered saline-MgCl ₂ (adding 0.1g of MgCl ₂ to PBS-Mg: PBS to a final concentration of 0.1M) to Nunc-Immuno Maxisorp ^TM 96 well microplate (NalgeNuncInternational, 100 μl was dispensed into each well of Rochester, NY, USA and left at 30 ° C. for 2 hours, followed by drying at 45 ° C. Probe DNA was prepared using genomic DNA of YC6002 strain. 10 μl of DNA and the same amount of photobiotin (Sigma, Madison, WI, USA) were mixed and chemically labeled for 30 minutes on a 400W low-pressure mercury lamp, followed by the addition of 1-buthanol. The excess photobiotin was then transferred to 1-buthanol layer for removal. The hybridization reaction was carried out with 200 μl of pre-hybridization solution [1 × 20 × SSC, 0.5ml of 100 × Denhardt's solution, 0.1ml of 10mg / ml heat denatured salmon DNA (Sigma), 5.0ml of formamide, 3.4ml of DW]. Blocking was performed by keeping at 37 ° C. for 10 minutes. 100 μl of a hybridization solution [pre-hybridization solution + Dextran sulfate 0.25 g] containing a probe per well was added and maintained at 49 ° C. for 3 hours, followed by hybridization reaction. Plates were washed three times with 1 × SSC, and 100 μl of streptoabidin-β-galactosidase (Sigma) solution was dispensed into each well and maintained at 37 ° C. for 30 minutes. After the reaction was completed, water was removed, and the wells were washed three times with 300 μl of 1 × SSC, and 100 μl of 4-methylumberilliferyl -D -galatopyranoside (4MUF, Sigma) was added thereto as a substrate. Absorbance was measured in Fluoroskan Fluorescence Plate Reader (MTX Lab Systems, Vienna, VA, USA) for a period of time. The measurements were analyzed using Microsoft Excel 2003. The determination of the results was determined to be homogeneous when the DNA-DNA reassociation values between the comparative strains were 70% or more or the thermal stability (ΔTm) was 5 ° C or lower, and the optimum renaturation temperature (TOR) was determined according to the following equation.

[Equation 1]

TOR = [0.41 × (G + C content) + 69.3] -45 (50% formamide)

Genomic DNA of the Asthrobacter sp. Strain compared to YC6002 was extracted and used for DNA-DNA hybridization. The hybridization temperature of YC6002 with G + C content of 61.2 mol% was 49 ° C. In Table 7, the homology of YC6002 and the comparative strains was 55, 42, 27, 20% and less than 70%, respectively, indicating that the endogenous bacterium YC6002 was a new species of the genus Arthrobacter sp.

Table 7 DNA-DNA homology levels between Arthrobacter species similar to endogenous bacterial YC6002 strain

Therefore, on the basis of physiological biochemical experiments and molecular biological experiments, this strain was identified as a new species of the genus Asthrobacter sp.

Identified as a New Species of the Arthrobacter sp. As of March 19, 2008, YC6002 The accession number KCTC 18127P was assigned by the Korean Collection for Type Cultures (KCTC).

Example 2 Production of Germination Inhibitors of Endogenous Bacteria YC6002

1. Selection of production medium of seedless germination inhibitor

In order to select a medium suitable for production of seedless germination inhibitors of the isolated strains, an experiment was performed by selecting five media (Table 8).

[Table 8] Medium components for production of seed germination inhibitors of endogenous bacterial YC6002 strain

YC6002 strain was inoculated into 500 ml of each selected medium, followed by incubation at 30 ° C. and 180 rpm for 72 hours, followed by centrifugation (Beckamn J-2HS, 7000 rpm, 15 min) to recover only the supernatant. In order to investigate the effect of suppressing seed germination with the collected supernatant, put 500 ㎕ of YC6002 culture supernatant on a petri plate with a sterile × 2 cm sterile dried kitchen towel, and then dispense the seeds of Radish (Cheongdae Bomba, Nongwoo Bio) Five repetitions were placed in three repetitions, and germination rates were examined after 25 ° C. and 72 h. Among them, the best seed germination inhibitor was selected and used as a mass production medium for structure determination of seed germination inhibitor.

In the selection of the medium for the production of the material with 0.1 TSB and 4 medium, the highest germination inhibitory effect was observed in 0.1 TSB, and in Czapek-dox broth medium, it was more inhibited than the control distilled water but less than 0.1 TSB (FIG. 4). Therefore, 0.1 TSB was used for mass production of seed germination inhibitor.

2. Production of seed germination inhibitors according to culture temperature

 Inoculated with YC6002 strain in 0.1 TSB selected as seed germination inhibitor production medium, and incubated at 20 ℃, 25 ℃, 30 and 180 rpm for 3 days to examine the germination inhibitor activity according to temperature, At intervals of time thereafter, samples were taken at 12-hour intervals, and the germination density was investigated in the same manner as above for seed germination inhibition.

After shaking culture for 72 hours at 20 ℃, 25 ℃, 30 ℃ was observed the results. YC6002 had the highest density at 30 ° C. and 24 h culture, and then gradually decreased, and also decreased pH until 12 h, but continued to increase thereafter (FIG. 5). Germination rate results by culture temperature showed that the culture medium and its supernatant showed a clear inhibitory effect compared to the control, the difference according to the culture temperature was not large (Fig. 6).

As a result of investigating inhibitor production by bacterial culture period, the germination inhibition effect was good in the culture solution and supernatant of YC6002 cultured for 48h under 30 ℃ temperature condition. As a result of investigating the germination inhibitory effect by period, it showed a definite germination inhibitory effect compared to the control after 1 day (Fig. 7).

Example 3: Preparation of a Microbial Formulation Containing a Strain of the Present Invention or Cultures thereof

In order for YC6002 strains to survive in natural environmental ecosystems or plants, and to suppress seed germination and growth, it is necessary to maintain nutrient supply (nutrients available in the short and medium to long term), protect bacteria, increase germination rate, and maintain short-term growth. Do. For this purpose, a bio-matrix was prepared. Biomatrices are made from a mixture of plant biopolymers, nutrients, supplements, and additives. Potato powder and soybean powder were used as plant material polymer materials, and glucose, peptone and inorganic salts were used as nutrients. As an auxiliary material, vermiculite or rice husk powder was used. As the additive, an extender (feldspar quartz mixed mineral, zeolite, etc.), a UV blocking agent, and a surfactant were used. Table 9 shows the composition components and the component amounts for producing the biomatrix.

TABLE 9 Bio Matrix Composition

1. Preparation of granules and hydrates

The YC6002 strain of the present invention was cultured in a fermenter (1 ton or more), or the cells obtained after centrifugation were concentrated and mixed with an aqueous 1% carboxymethyl cellulose (CMC) solution to form a film. The prepared biomatrix was uniformly mixed in a homogenizer such that the number of encapsulated YC6002 cells was about 10 ⁷ -10 ⁸ per gram of the biomatrix. The YC6002 strain and the biomatrix complex thus prepared were dried, dried and uniformly pulverized to prepare pellets.

2. Preparation of liquids (suspensions)

Cell cultures obtained by culturing the YC6002 strain in a large fermenter (1 ton or more) or cells obtained by centrifugation were mixed with feldspar quartz mixed mineral 1:10, respectively, and prepared as a suspension.

Example 4: Field weed germination inhibition of the microorganism preparation containing the strain of the present invention or its culture

The YC6002 strain culture medium cultured by the method of Example 2 was treated with 50 ml per cow pot seeded with major weeds, and the following results were obtained. The test method was a method developed by the Korea Research Institute of Chemical Technology (located in Yuseong-gu, Daejeon). Filling the soil with 350 cm ² square plastic pots for the cultivation of plants, 5 crops (corn, soybean, cotton, wheat, rice) and 10 weeds (flower and weed varieties: corn, dolphin, wheat, barley, American Opening Fields: Five broad-leaved weeds-Crown, Korean herbaceous plants, Sparrows, Doktors, Buckwheat flowers) were divided and sown, and managed under greenhouse conditions. One day after seeding, spraying was carried out using a spray gun (IWATA, Wider-61) in the amount of 200, 100, 50 ml / 2 pots of strain culture medium, and then irradiated with a tube after 14 days in a greenhouse condition. In the second test, all the control including the seeding was carried out in the same manner as the first test, and the treated microorganism suspension was irrigated in the pot in the amount of 400, 200, 100 ml / 2 pot.

In the primary treatment range of the culture medium containing the YC6002 strain, there was no inhibitory effect on 5 crops and 10 weeds, but 400ml was treated in the second test in which the bacterial culture was concentrated and formulated with minerals to increase the amount of treatment. When some herbicides were confirmed herbicidal effect, the external manifestations were growth inhibition (Table 10, Figure 8). As a result, the microbial agent formulated with the YC6002 strain is most effective to be used as a non-selective herbicide sown in the field before the seeds of the plant germinate.

Table 10. Effect of YC5480 strain microbial agent on the weed and crop germination

* 0; No effect, 10-30; Slight inhibition, 40-60; Moderate inhibition, 70-90; Severe inhibition, 100; Completely suppressed.

As described above, according to the present invention, the germ and growth inhibition of the seed containing the new strains Arthrobacter zoysiae YC 6002 and Asthrobacter zoysiae or its culture medium as an active ingredient Water pesticides can be prepared.

Biopesticides using the new strain Arthrobacter zoysiae of the present invention is excellent in inhibiting germination and growth of seeds, and has the effect of inhibiting seed germination and growth of weeds in the cultivation area, so that crops grow to some extent. There is an outstanding advantage that can help you grow continuously without being disturbed by newly generated weeds. These characteristics make it possible to develop as a non-selective herbicide, which occupies more than half of the domestic and international herbicide market, and in particular, it is more safe than chemical synthetic herbicides.

1 is a scanning electron micrograph of the endogenous bacteria YC6002 strain of the present invention.

Figure 2 is a photograph showing the phylogenetic tree of the endogenous bacteria YC6002 strain of the present invention.

Figure 3 is a two-dimensional thin layer chromatography photograph of the endogenous bacteria YC6002 cell phospholipids of the present invention.

Figure 4 is a graph showing germination and growth inhibition of seedless using the culture medium and supernatant of the endogenous bacteria YC6002 strain of the present invention.

5 is a graph showing the population density and pH change of YC6002 by shaking culture period at 0.1 TSB, 30 ° C., and 180 rpm of the endogenous bacterial YC6002 strain of the present invention.

Figure 6 is a graph showing the germ-free germination inhibition effect of the endogenous bacteria YC6002 strain of the present invention at the temperature-specific conditions.

Figure 7a is a graph showing the germination of seed-free germination of the culture medium by varying the incubation period of the endogenous bacteria YC6002 in 0.1TSB medium of the present invention.

7B is a graph showing the germination of seed-free germination of supernatant by varying the incubation period of the endogenous bacteria YC6002 in 0.1TSB medium of the present invention.

8 is a photograph showing the inhibitory effect by treating the endogenous bacteria YC6002 of the present invention in 0.1TSB medium and centrifugation to obtain the cells, and then the suspension is treated to major crops and field weeds (left: untreated, right: treated)

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY          Jgreen Inc. <120> Biotic pesticide using Arthrobacter zoysiae with inhibition          activity of germinaton and growth of seeds <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1390 <212> DNA <213> Arthrobacter zoysiae <400> 1 atgcagtcga acgatgaccc ggtgacttgc accggtgatt agtggcgaac gggtgagtaa 60 cacgtgagta acctgccctt aactctggga taagcctggg aaactgggtc taataccgga 120 tatgactcct catcgcatgg tggggggtgg aaagctttat tgtggttttg gatggactcg 180 cggcctatca gcttgttggt gaggtaatgg ctcaccaagg cgacgacggg tagccggcct 240 gagagggtga ccggccacac tgggactgag acacggccca gactcctacg ggaggcagca 300 gtggggaata ttgcacaatg ggcgcaagcc tgatgcagcg acgccgcgtg agggatgacg 360 gccttcgggt tgtaaacctc tttcagtagg gaagaagcga aagtgacggt acctgcagaa 420 gaagcgccgg ctaactacgt gccagcagcc gcggtaatac gtagggcgca agcgttatcc 480 cggaattatt gggcgtaaag agctcgtagg cggtttgtcg cgtctgccgt gaaagtccgg 540 ggctcaactc cggatctgcg gtgggtacgg gcagactaga gtgatgtagg ggagactgga 600 attcctggtg tagcggtgaa atgcgcagat atcaggagga acaccgatgg cgaaggcagg 660 tctctgggca ttaactgacg ctgaggagcg aaagcatggg gagcgaacag gattagatac 720 cctggtagtc catgccgtaa acgttgggca ctaggtgtgg gggacattcc acgttttccg 780 cgccgtagct aacgcattaa gtgccccgcc tggggagtac ggccgcaagg ctaaaactca 840 aaggaattga cgggggcccg cacaagcggc ggagcatgcg gattaattcg atgcaacgcg 900 aagaacctta ccaaggcttg acatgggccg gaccgggctg gaaacagtcc ttcccctttg 960 gggccggttc acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa 1020 gtcccgcaac gagcgcaacc ctcgttccat gttgccagcg cgtaatggcg gggactcatg 1080 ggagactgcc ggggtcaact cggaggaagg tggggacgac gtcaaatcat catgcccctt 1140 atgtcttggg cttcacgcat gctacaatgg ccggtacaaa gggttgcgat actgtgaggt 1200 ggagctaatc ccaaaaagcc ggtctcagtt cggattgggg tctgcaactc gaccccatga 1260 agtcggagtc gctagtaatc gcagatcagc aacgctgcgg tgaatacgtt cccgggcctt 1320 gtacacaccg cccgtcaagt cacgaaagtt ggtaacaccc gaagccggtg gtacgtaacc 1380 ccttgtcgga 1390  

Claims (3)

Agrobacterium for inhibiting germination and growth of seeds, which comprises Arthrobacter sp. YC6002 strain (Accession No .: KCTC 18127P) or its culture as an active ingredient. delete The germination and growth inhibition of seeds according to claim 1, wherein the Arthrobacter sp. YC6002 strain (Accession No .: KCTC 18127P) or its culture is mixed with at least one of starch, crude protein, and rock meal. Dragon bio pesticides.

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