KR101199931B1 - Promotion of nitrogen mineralization of organic fertilizers and control of plant diseases using bacillus velezensis KRICT934 - Google Patents

Abstract

The present invention relates to a microbial agent containing the Bacillus Velensis KRICT934 strain and its culture, and to a method for controlling plant diseases using the same. The Bacillus Velensis KRICT934 strain according to the present invention has excellent antibacterial activity against various plant diseases. At the same time, the activity of promoting the nitrogen mineralization of oil cakes is very good, and the economical substitution effect of the existing microorganisms is large and has a high performance, thereby increasing the marketability and productivity of crops.

Description

Promotion of nitrogen mineralization of organic fertilizers and control of plant diseases using bacillus velezensis KRICT934}

The present invention relates to a method for promoting nitrogen atomization and controlling plant diseases of organic fertilizers using Bacillus Velensis KRICT934 strain, and more particularly, a microbial agent containing the novel Bacillus Velensis KRICT934 strain and its culture and It relates to a plant disease control method.

Agricultural productivity has been steadily increasing due to the use of synthetic pesticides and chemical fertilizers and technological improvements in cultivation methods.However, the use of synthetic pesticides and chemical fertilizers has contaminated soil, water and agricultural products, toxicity, disturbance of ecosystems, and emergence of resistant strains. Various problems are raised, and in order to reduce their usage according to consumers' preference for high-quality agricultural products, various countries including Korea are implementing policies. Alternative technologies for these chemical fertilizers and synthetic pesticides are organic fertilizers and biopesticides.

Organic fertilizers improve the physicochemical and biologic properties of soil by supplying organic matter to the soil to increase nutrient and moisture retention and soil stiffness (Sim Gil-gil et al. 1992. Hantoji 25 (1): 52-56; The Korean Journal of Organic Waste Science 7 (1): 23-30), Organic fertilizers themselves are a source of nutrients, which is a medium for soil microorganisms to increase crop growth stability and soil productivity (Lee Kyungja et al., 2004. 2): 145-149).

In addition, humic acid and pyruvic acid produced from organic matters sometimes have the effect of promoting plant growth (Yoo Sung-jun et al., 1996. Hantobuji 29 (3): 297-302). Currently, oil cake is the most used organic fertilizer. It has been widely used as fertilizer because it has high starch and protein content and is not only used for livestock feed but also has high organic matter and nitrogen content and also contains phosphoric acid and potassium. In Korea, about 10 kinds of oil foils are registered, such as sesame jelly, chaejongbak, cottonseed gourd, castor gourd and skim steel.

However, organic fertilizers, unlike composting, do not go through the maturation process, which can cause gas damage by temporary decomposition when applied to the soil, so that crops must be grown after a stabilization period of about two weeks or more. However, in the case of farmers, the period between the previous work and the marquise is short, and the gas damage occurs frequently due to the lack of recognition. In addition, the existing products are not able to supply the nitrogen source necessary for the growth of seedlings quickly after planting, and the initial growth is poor. Especially in the case of water, the growth is significantly lower than other fermented organic fertilizers such as compost.

Therefore, in order to reduce the period of staying for two weeks and to supply high quality organic fertilizers, it is possible to accelerate the mineralization by early decomposition when used in soil, and then to organic nitrogen (ammonia nitrogen = NH ₄ -N, nitrate nitrogen = NO ₃ -N). There is a great need to solubilize nutrients with conversion. This protein degradation and nitrogen mineralization process is mainly carried out by microorganisms. Several different types of microorganisms, such as bacteria, filamentous fungi and actinomycetes, liberate ammonium from organic nitrogen compounds.

On the other hand, in the land using organic fertilizers, since the fertilizer is mainly used for producing organic products, synthetic pesticides cannot be used to control various plant diseases. Biopesticides should be used to control these plant diseases. Biopesticides are largely divided into plants, microorganisms, natural enemies, natural bioactive substances and genetically modified organisms (GMOs). Research on the development of biopesticides, especially microbial fungicides, has been ongoing as a major field of plant pathology for the last 70 years, and over 40 products have been developed over the past decade.

The Gram-positive bacterium, Bacillus, is the second most widely researched and commercialized bacterium after Pseudomonas , producing a large number of secondary metabolites that exist in soil in a large number and are biologically active, endogenous to heat and stable to harsh environments. Has the characteristic of forming spores (endospore). Bacillus velogenis strain having such characteristics was developed by Gustafson in 1994 under the trade name Kodiak as a seed and furrow treatment agent of cotton and peanuts (Backman et al., Improving Plant Productivity with Rhizosphere Bacteria, pp. 3-8).

In addition, in 2001, Bacillus bellenosis bacteria and Bacillus amyloliquipecission ( Bacillus amyloliquefaciens) was mixed with the bacteria releases a microbial fungicide called bio-Ile (BioYield). In China, many bacteria of the genus Bacillus are widely used as bacteria for improving yields. Bayer, Germany, has developed a soil infection control agent using Bacillus FZB 24. In addition, Taensa in the United States has released a microbial fungicide using the same bacteria. AgraQuest in the United States has developed a microbial fungicide using a strain of Bacillus Belegensis QST713 called Serenade.

In Korea, research on biological control of ginseng root rot from the early 1970s has been conducted on biological control of pepper blight, cucumber and strawberry wilted disease, sesame mozzarella and ash fungus, but the effects of instability, lack of formulation, and economic feasibility There is no great progress due to the back.

Until now, as a microbial disinfectant using microorganisms in Bacillus, products such as 'terrace', 'hole in one', 'greenol', 'sealus', 'shooting star' and 'jaetan' are registered. Therefore, it has been shown to be effective in powdery mildew and gray mold, grass brown leaf blight or grass pistil blight, and so many restrictions are placed on its use.

However, there are many reports of microorganisms that promote the process of inorganic fertilization of organic fertilizers (Kim Jin-ho and Joo Gil-jae, 2008. Korean Patent Application No. 2008-0008928) or microorganisms that are effective against plant diseases. At the same time, there are no reports of microorganisms owned.

Therefore, the present inventors have tried to select microorganisms that reduce the period of stabilization of organic fertilizers as well as reduce various plant diseases by promoting the initial proteolysis when using organic fertilizers to increase the production of inorganic nitrogen, thereby simultaneously performing the above two functions. After screening the microorganisms owned, the control effect on various plant diseases and the inorganic nitrification promoting activity of the excellent organic matter was confirmed, and completed the present invention.

An object of the present invention is to promote the initial proteolysis of organic fertilizers and increase the production of inorganic nitrogen to shorten the stabilization period of organic fertilizers as well as to reduce various plant diseases and microbial preparations containing them and their cultures It is to provide a method for controlling plant diseases.

The present invention, in order to achieve the above object, provides a microbial agent containing a novel Bacillus Velensis KRICT934 strain and its culture and a plant disease control method using the same.

The novel Bacillus Velensis KRICT934 strain of the present invention is isolated from the roots of Chinese cabbage, and is characterized by having both inorganic nitrogenization promoting activity of organic matter and antibacterial activity against plant pathogens.

Bacillus bellensis of the present invention ( Bacillus velezensis ) KRICT934 (Accession No .: KCTC 11774BP) Strain and Bacillus Velensis ( Bacillus velezensis ) The culture of KRICT934 (Accession Number: KCTC 11774BP) strain is 1 selected from rice blast, rice leaf blight, tomato ash mold, tomato blight, wheat rust disease, barley powdery mildew, pepper anthrax, radish blight and pepper blight. It is characterized by having a strong antagonistic action against plant diseases of more than one species.

Hereinafter, the present invention will be described in detail.

The present invention provides a Bacillus velezensis KRICT934 (Accession No .: KCTC 11774BP) strain and its culture, which are separated from the root of Chinese cabbage and have both an inorganic mineralization promoting activity and an antibacterial activity against plant pathogens. do.

More specifically, Bacillus bellensis ( Bacillus) according to the present invention velezensis ) KRICT934 strains were isolated from the roots of Chinese cabbage, and their morphological and biochemical characteristics were as follows.

Tryptic soy agar medium (Tryptic soy agar) is spreading thinly, the single colony has a small snowflake shape, rod-shaped endogenous spores were confirmed to be positive in Gram staining. In addition, dextrin, L-arabinose, cellobiose, D-fructose, gentobiose, α-D-glucos (α-D- glucose, D-mannose, D-ribose, D-xylose, glycogen, methyl α-D-glycoside ), D-raffinose, D-Turanose, etc. to generate acid and hydrolyze tween 40 and tween 80. there was. See FIG. 5.

In addition, the base sequence (SEQ ID NO: 1) and the base sequence of the gyrA gene (SEQ ID NO: 2) of the 16S rRNA gene of the Bacillus Velensis KRICT934 strain were confirmed to be Bacillus velezensis . Bacillus velezensis ) was named KRICT934 and deposited with the Korea Research Institute of Bioscience and Biotechnology on October 4, 2010 and was assigned the accession number KCTC 11774BP. See FIGS. 1 to 4.

In addition, it was confirmed that the culture of the Bacillus Velensis KRICT934 strain or the Bacillus Velensis KRICT934 strain of the present invention has the ability to promote nitrogen atomization of mixed milk.

More specifically, as a result of investigating the nitrogen atomization-promoting activity of mixed Bacillus spp. Against Bacillus Velensis KRICT934 strains according to the ratio of degreasing steel and vermiculite, the nitrogen-mineralization activity of Bacillus spp. It was confirmed that it was excellent. See Figures 6-8.

The present invention has the above characteristics Bacillus Velensis ( Bacillus velezensis ) containing the culture of the KRICT934 (Accession No .: KCTC 11774BP) strain or Bacillus velezensis KRICT934 (Accession No .: KCTC 11774BP) strain, to promote the nitrogen inorganicization of organic matter and antibacterial activity against plant pathogens It provides a microbial agent having at the same time.

The microbial agent has a strong antagonistic action against at least one plant disease selected from rice blast, rice leaf blight, tomato gray mold, tomato late blight, wheat red rust, barley flour, pepper anthrax, radish blight and pepper blight. It is characterized by.

The present invention is the Bacillus Velensis ( Bacillus velezensis ) KRICT934 (Accession No .: KCTC 11774BP) strain or the Bacillus Velensis ( Bacillus velezensis ) Plants characterized by treating microorganisms containing cultures of strains of KRICT934 (Accession No .: KCTC 11774BP) simultaneously with the microorganism preparation having an antimicrobial activity against nitrogen pathogenicity of plants and antimicrobial activity against plant pathogens on the ground or in the field of crops. Provide a method for controlling the bottle.

The plant disease is at least one selected from rice blast, rice leaf blight, tomato ash mold, tomato blight, wheat red rust, barley flour, pepper anthrax, radish blight and pepper blight, applied to crops in a conventional manner. do.

In a specific method, the microbial preparation according to the present invention is sprayed onto soil, crop leaves, stems, flowers or fruits, and can be used by diluting the microbial preparation in water, in order to add the antimicrobial activity of the microbial strain of the present invention. It may further include foliar spray or irrigation spray. In addition, during the spraying season, it is possible to further increase the control effect by prophylactically treating each plant pathogen before the onset and propagation.

In addition, the present invention is the Bacillus Velensis ( Bacillus velezensis ) An organic fertilizer containing a culture of KRICT934 (Accession No .: KCTC 11774BP) strain or Bacillus velezensis KRICT934 (Accession No .: KCTC 11774BP) strain may be provided.

Bacillus Velensis KRICT934 strain according to the present invention has an excellent antimicrobial activity against various plant diseases, and at the same time has a very good activity of promoting the nitrogen mineralization of oil foil, has a high economic replacement effect for the existing microorganisms and has a high performance This has the effect of increasing the marketability and productivity of crops.

Figure 1 shows the 16S rRNA gene sequence of the KRICT934 strain according to the present invention,
Figure 2 shows the gyr A gene sequence of the KRICT934 strain according to the present invention,
Figure 3 shows the phylogenetic tree by the 16S rRNA gene sequence of the KRICT934 strain according to the present invention,
Figure 4 shows the phylogenetic tree by the gyr A gene sequence of the KRICT934 strain according to the present invention,
5 is a result of examining the enzyme production capacity of the solid culture extract of KRICT934 strain according to the present invention,
(A: protease, B: chitinase, C: cellulase)
6 is a result of investigating the nitrogen mineralization promoting activity in mixed oil foil for the KRICT934 strain of the present invention according to the ratio of skim steel and vermiculite (degreasing steel: vermiculite = 2: 8, v / v),
7 is a result of investigating the nitrogen mineralization promoting activity in mixed oil foil for the KRICT934 strain of the present invention according to the ratio of skim steel and vermiculite (degreasing steel: vermiculum = 1: 1, v / v),
FIG. 8 shows the results of investigating the activity of nitrogen nitrification in soil of the culture of the KRICT934 strain of the present invention cultured from skim and vermiculite (degreasing steel: vermiculite = 1: 1, v / v).
9 is a result of examining the mycelial growth inhibitory activity of KRICT934 strain according to the present invention against various phytopathogens,
10 is a result of examining the preventive effect of the treatment of the culture of the KRICT934 strain according to the present invention one day before inoculation for various plant diseases,
(RCB: Rice Blast, RSB: Rice Leaf Blight, TGM: Tomato Ash Mold, TLB: Tomato Blight, WLR: Wheat Red Rust, BPM: Barley Powder, RPA: Chilli Anthrax)
11 is a result of examining the control effect of the KRICT934 strain solid culture medium according to the present invention against the withering disease,
(Left port: no treatment port, right port: KRICT934 treatment port)
12 is a result of examining the control effect of KRICT934 strain solid culture medium according to the present invention against pepper blight.
(Left port: KRICT934 treatment port, right port: no treatment port)

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by the following examples, and various modifications or changes can be made within the spirit and scope of the present invention to those skilled in the art.

Hereinafter, the technical and scientific terms used herein will be understood by those skilled in the art without departing from the scope of the present invention. Descriptions of known functions and configurations that may be unnecessarily blurred are omitted.

[ Example  One] Bacillus Velensis KRICT934  Isolation of strain

Various types of healthy crop roots such as cabbage, tomato and radish were divided into small pieces, soaked in 1% NaOCl for 1 minute, and then washed in sterile water for 1 minute. The root tissue was ground by adding 0.1 M MgSO ₄ solution and then plated onto potato dextrose agar (PDA; Difco, USA). After culturing at 30 ° C., when bacterial colonies appeared, single colonies were separated by three to four passages in nutrient agar medium (Nutrient agar).

A total of 504 strains could be isolated from the isolated single colony, and the proteolytic enzyme-producing ability and nitrogen-mineralization-promoting activity of the oil foil were finally examined by the same method as in Examples 3 and 4, and finally the most excellent activity from the cabbage roots. Strains were selected and named KRICT934.

[ Example  2] Bacillus Velensis KRICT934  Identification of the strain

Identification of the Bacillus Velensis KRICT934 strain selected in Example 1 was performed using the morphological / biochemical characteristics and 16S rRNA gene and gyr A gene sequence analysis.

(1) Morphological and biochemical properties

The Bacillus Velensis KRICT934 strain selected in Example 1 was spread out thinly on Tryptic soy broth (Tryptic soy broth), a single colony was confirmed to have a small snowflake shape, the selection strain is rod, It was positive in Gram staining. Forms endogenous spores, dextrin, L-arabinose, cellobiose, D-fructose, gentobiose, α-D-glucos ( α-D-glucose, D-mannose, D-ribose, D-xylose, Glycogen, methyl α-D-glycoside -D-glycoside, D-raffinose, D-Turanose, etc. are used to generate acid and hydrolyze tween 40 and tween 80. Appeared. When comparing these characteristics with the standard strain (type strain) LMG22478, there were various differences as shown in Table 1 below.

(2) DNA sequence analysis

As a result of analyzing the nucleotide sequence (SEQ ID NO: 1) of the 16S rRNA gene of the Bacillus Velensis KRICT934 strain selected in Example 1, Bacillus subtilis complex ( Bacillus) as shown in FIG. subtilis complex).

For more accurate identification, sequencing of the gyr A gene was performed. As a result, the base sequence (SEQ ID NO: 2) of the gyr A gene was analyzed. As shown in FIG. 4, the strain selected in Example 1 exhibited about 98.5% homology with Bacillus bellensis LMG22478T. Could.

The strain obtained in the present invention from the above results was identified as Bacillus Velensis strain, named the strain Bacillus Velensis KRICT934 and deposited on October 4, 2010 to the Korea Biotechnology Research Institute microbial resource center No. No. KCTC 11774BP.

[ Example  3] Bacillus Velensis KRICT934  Enzyme of strain Generation  Research

The KRICT934 strains were subjected to bioassay for protease, cellulase, and chitinase to investigate the protein resolution and the production of other enzymes.

1/10 Luria-Bertani agar (purchased by Difco, USA) containing 1% (w / v) skim milk (purchased by Difco, USA) for protease production KRICT934 strains were inoculated with a stroke using a loop in the center of the medium, and after incubating at 30 ° C. for 24 hours, the radius of the clear zone around the colonies was measured.

Chitin agar medium (Colloidal chitin 7 g, KH ₂ PO ₄ 3 g, K ₂ HPO ₄ 2 g, NH ₄ Cl 1 g, MgSO ₄ 7H ₂ O 0.2 g, CaCl ₂ ? 2H ₂ O 0.01 g, yeast extract 0.1 g, Bacto agar 15 g, distilled water 1 L), and inoculated with KRICT934 strain in the center of the medium by incubation for 3 days at 30 ℃ to investigate the radius of the clear zone around the colony It was.

In addition, cellulose agar medium (MgSO ₄ ~ 7H ₂ O 0.1 g, CaCl ₂ ~ 2H ₂ O 0.2 g, KH ₂ PO ₄ 0.3 g, K ₂ HPO ₄ 0.5 g, carboxymethylcellulose 5 g, yeast extract 0.1 g, Bacto agar 15 g, distilled water 1 L), inoculated with KRICT934 strain in the center of the medium incubated for 3 days at 30 ° C, and then poured 1 mg / ml congo red on the medium. Shake well for a minute, discard, and march three times with 1 M NaCl to investigate the size of the clear zone formed around the colonies.

As a result, as shown in Table 2, the KRICT934 strain produced proteases and cellulase, but it was confirmed that the chitinase does not produce.

On the other hand, in order to investigate the production of these enzymes in the solid culture of the Bacillus Velensis KRICT934 strain, sterile skim: Vermiculite (2: 8, v / v) medium (50 g; water) in Petri dish. 17.5 ml) was inoculated with 5 ml of the culture medium incubated for one day at 30 ° C. and 150 rpm in a tryptic soy liquid medium. After inoculation, the cells were incubated at 30 ° C. for 3 days, and the media were shaken every day. 10 ml of distilled water was added thereto for 5 g of the solid culture thus cultured, and then sonicated for 10 minutes.

After filtration and sterilization using a 0.2 μm membrane, 35 μl of the obtained extract was treated with a sterile paper disc and then subjected to three enzyme assay media. Proteases were observed after one day, and cellulase and chitinase were examined for the formation of clear zones after three days.

As a result, as shown in Figure 5 Bacillus Velensis KRICT934 strain was confirmed to produce a lot of proteases and cellulase even in a solid culture.

[ Example  4] Bacillus Velensis KRICT934  Strain Foil  Nitrogen inorganicization promoting activity

In order to investigate the nitrogen-inorganic activity of the oil of the selected Bacillus Velensis KRICT934 strain, a mixture containing sterile skim: Vermiculite (2: 8, v / v) in a Erlenmeyer flask (50 g; water; 17.5 ml) was inoculated with 5 ml of a culture of selected Bacillus velogenis KRICT934 strain incubated at Tryptic soy broth for 30 days at 150 rpm for 1 day.

After the inoculation, the cells were incubated at 30 ° C. for 3 days, and the media were shaken every day. To this culture (1.5 g) was mixed mixed milk (nitrogen content = 4.2%) (1.5 g) and then 1 ml of water was added. After incubating at 30 ° C., samples were collected with 2M KCl on the 5th, 7th, 9th, and 11th days, and the amount of inorganic nitrogen was measured using Kjeldahl distillation. At this time, the control group was irradiated with control group 1 mixed with oil foil (1.5 g) + distilled water (1 ml) and control group 2 treated with culture (1.5 g) alone.

In addition, in order to determine the degree of nitrogen mineralization activity of milk gourd at different ratios of skimmed steel and vermiculite of solid medium, Bacillus velensis KRICT934 strain was incubated in skimmed steel: vermiculite (1: 1, v / v) for 3 days, When the culture medium was treated with oil, the degree of nitrogen mineralization in the laboratory was examined, and all other methods were performed as described above.

As a result, as can be seen in Figures 6 and 7, it was confirmed that the selected Bacillus Velensis KRICT934 strain of the present invention promotes the nitrogen inorganicization of oil foil.

On the other hand, the degree of oily nitrogen mineralization activity in the soil for the culture culture in skim steel: vermiculite (1; 1, v / v) medium was measured. 1 g of mixed oil foil and 1 g of culture medium were mixed with 10 g of soil, and 2 ml of distilled water was added thereto. As a control, control 1 containing soil (10 g) + oil foil (1 g) + distilled water (3 ml) and control 2 with soil (10 g) + culture (1 g) + distilled water (3 ml) were investigated. It was.

As a result, as can be seen in Figure 8, it was confirmed that the selected Bacillus Velensis KRICT934 strain of the present invention has a very strong activity for inorganic nitrogenization of organic nitrogen of oil foil in the soil.

[ Example  5] Bacillus Velensis KRICT934  Strain Plant pathogen  Growth inhibitory activity

Nine phytopathogens were used to investigate the effects of the selected Bacillus velogenis KRICT934 strain on the growth of various phytopathogens.

In the middle of the PDA medium was inoculated with mycelium pieces obtained from the culture of the phytopathogenic bacteria growing. Streaking the selected strains of KRICT934 in a straight line about 2.5 cm and then incubated at 25 ℃, the growth inhibition of phytopathogens by the KRICT934 strain was measured when the phytopathogens grow to the edge.

As a result, as can be seen in Table 3 and Figure 9 it was confirmed that strongly inhibit the growth for most phytopathogens.

[ Example  6] Inoculation 1 day before On foliar spray  7 kinds by In plant diseases  Preventive effect

The preventive effect of the treatment of the culture of the KRICT934 strain according to the present invention one day before the inoculation of various plant diseases was investigated.

The various plant diseases were tested against seven kinds of plant diseases including rice blast, rice leaf blight, tomato ash mold, tomato blight, wheat red rust, barley flour, pepper anthrax.

First, the Bacillus velogenis KRICT934 strain was inoculated into a sterile 50 ml Tryptic soy broth (purchased by Becton and Dickinson) and then incubated at 150 rpm for 3 days at 30 ° C. Tween 20 was added to a concentration of 250 µg / ml in a 40 ml dilution solution diluted three-fold with distilled water, and then treated with rice, tomatoes, wheat, and barley, respectively, and dried at room temperature. It was left for 1 day.

In the case of rice, Tween 20 was pre-spreaded with a solution of 250 µg / ml 4 hours before the culture was sprayed in order to enhance the electrodeposition power, and then dried at room temperature. The rice, tomatoes, wheat and barley used in the experiment were filled with water or soil for horticulture and 70% in plastic pots with a diameter of 4.5 ㎝, and then seeded and grown in a greenhouse at 25 ± 5 ° C. for 1 to 4 weeks.

In case of rice blasting , spray seedlings of spore suspension (5 × 10 ⁵ spores / ml) of Magnaporthe oryzae (Korea Research Institute of Chemical Technology), which is the causative agent of blasting disease, are spray-inoculated on seedlings of 3-4 leaf stages After a day of wet treatment, the incubation was induced by incubation for 5 days in a constant temperature room at 25 ℃.

In the case of rice leaf blight, five-leaf seedlings were inoculated with the culture obtained by incubating Rhizoctonia solani , the causative bacterium, in a medium (90 g of wheat bran, 15 g of chaff and 100 ml of distilled water) for 7 days. After 4 days of wet treatment on a 25 ° C. wet room, 4 days of incubation in a 25 ° C. constant temperature room induced the onset.

In case of tomato late blight, Phytophthora , which is the causative agent of late blight in 3-4 leaf tomato seedlings Inoculation of the influenza suspension extracted from the jujube sac (5 × 10 ⁴ sporangia / ml) of infestans , Kangnung National University), followed by 2 days of wet treatment in a 25 ° C. chamber and 1 day incubation at 25 ° C. It was.

In case of tomato gray mold disease, Botrytis , a causative agent of gray mold disease in tomato seedlings of 3-4 leaves After treatment with spore suspension (5 × 10 ⁵ spores / ml) of cinerea (Korea Research Institute of Chemical Technology), it was incubated for 3 days in a 20 ° C wet room to induce the onset.

In the case of wheat rust, Puccinia , a causative agent of rust disease known as a biotic parasite in the first leaf seedlings recondita (Incheon University) spores were sprayed in 250 ㎍ / mL Tween 20 solution in an amount of 0.67 g spores / ℓ, sprayed in a humidified environment at 20 ℃ for 1 day, and then transferred to a constant temperature chamber at 20 ℃. Incubation was induced by daily culture.

In case of barley powdery disease, Erysiphe , the causative agent of powdery mildew, passaged from host plants to barley seedlings, Erysiphe graminis f. sp. hordei , Korea Research Institute of Chemical Technology) and spores were inoculated and incubated for 7 days in a constant temperature room at 20 ℃ to induce the onset. The area ratios of rice blast, wheat rust, and barley powdery mildew were 7 days after inoculation, 8 days after inoculation for rice leaf blight, and 3 days after inoculation for tomato ash and tomato late blight.

On the other hand, for the control activity test for pepper anthrax, the seedlings of the most peppers are sown after filling 70% of gardening topsoil in a plastic pot with a diameter of 7.0 ㎝, and growing them in a greenhouse until 3 to 4 leaves, and then the Bacillus prepared above. Cultures of the Belizesis KRICT934 strain were foliarly sprayed. After spraying the sample was dried at room temperature for 24 hours, and then spray inoculated with a spore suspension (4 × 10 ⁵ spores / ml) of the pepper anthrax pathogen , Colletotrichum coccodes (Korea University). After 2 days of invasion in a humid room, it was left in a constant temperature room at 25 ° C. for 1 day, and the lesion area ratio was examined after 3 days of inoculation.

Using the lesion area ratio obtained from the above, the control value was calculated according to Equation 1 below.

As a result, as shown in Figure 10, the Bacillus Velensis KRICT934 strain of the present invention was confirmed to show a high antimicrobial activity against all seven plant diseases tested, in particular, excellent antibacterial against rice leaf blight blight and tomato blight It was confirmed to have activity.

[ Example  7] Bacillus Velensis KRICT934  Strains of radish Withered  For effect

In order to investigate the control effects of plant diseases when the solid cultures of Bacillus Velensis KRICT934 strains were treated with soil, the control effects against wilted ulcer disease were investigated.

Solid incubated at 30 ° C. for 3 days after inoculating the strain into skim steel: Vermiculite (2: 8, v / v) solid medium (50 g; 17.5 ml of water) in the same manner as in Example 3. Cultures were used as samples.

Garden seeds were sown in 1: 1 (v / v) soil mixed with horticultural soil and Vermiculite, and cultivated in a greenhouse for 14 days. The roots are pulled out and washed with water, and then the roots are removed from Fusarium Fusarium. oxysporum f. sp . raphani ) was immersed in a spore suspension (1.0 × 10 ⁷ spores / ml) for 30 minutes. Spore suspension was inoculated in malt extract broth (MEB) and incubated in a 25 ° C incubator for 7 days, followed by filtration of 4-ply gauze to remove mycelia and centrifugation of the harvested spore suspension (4 ° C). After dilution with sterile water to adjust the concentration. Solid cultures were applied to the roots of the soaked seedlings, and then transplanted using a horticultural soil containing 5% of the solid cultures in a 50 ml volume pot. The incidence was examined after 3 weeks in a 25 ℃ wet room and then transferred to a greenhouse.

Incidence was measured using the following index (index).

0: wholesome, 1: subterranean part browned, but above-ground decayed and without symptoms, 2: subterranean part browned and withered, 3: subterranean part browned, above-ground withered and yellowed, 4: subterranean part browned Terrestrial yellowing withered and deciduous, 5: dead.

As a result, as can be seen in Figure 11, the solid culture of the Bacillus Velensis KRICT934 strain of the present invention showed a 27% control against the disease withered.

[ Example  8] Bacillus Velensis KRICT934  Effect of Strain on Pepper Plague

The effect on pepper blight was investigated using the strain solid culture medium of the present invention used in Example 7.

Four-leafed nasal cayenne pepper seedlings were transferred to a pot of 200 ml volume, and the solid culture was treated on the pot surface with an amount corresponding to a pot volume ratio of 10% and grown in a greenhouse for one week. Pepper toxin ( Phytophthora capsici ) was inoculated in oatmeal agar (OMA) medium and incubated for one week at 25 ℃. One week later, the surface of the medium in which pepper blight was grown was scraped off with a brush to remove mycelia, and then incubated for 24 hours in a humid room at 25 ° C. Pour cold sterilized water at 4 ℃ into red pepper bacteriophage medium, and scrape it with a brush to recover the intestine sac, adjust the recovered saccharin to 3 × 10 ³ saccharin / ml, put it in the refrigerator, take it out after one hour, and remove it at room temperature for 20 minutes. Put it.

One week after the solid culture was treated, the pepper seedlings were transferred to a tray, and 10 ml of pepper seedlings of the above-mentioned pepper plague were irrigated in pepper seedlings. The trays were filled with water about 0.5 cm and wetted for 24 hours in a humid chamber (95% RH, 25 ° C.).

Incidence was measured using the following index (index).

0: wholesome, 1: the branch is soft and begins to brown, 2: the leaves are struck down with 30-50% of the disease, 3: the leaves are struck with the 50-70% of the disease , 4: 70-90% of all diseased leaves and stems except upper side, 5: dead.

As a result, as shown in Figure 12, it was confirmed that the solid culture of the Bacillus Velensis KRICT934 strain of the present invention showed a high control activity of 55% against pepper blight.

Korea Biotechnology Research Institute KCTC11774BP 20101004

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Claims (7)

Isolate from the roots of Chinese cabbage, one species selected from organic mineralization promoting activity and selected from rice blast, rice leaf blight, tomato blight, tomato blight, wheat red rust, barley flour, pepper anthrax, radish, and pepper blight Bacillus velezensis KRICT934 (Accession No .: KCTC 11774BP) strain having simultaneously antimicrobial activity against the causative bacteria. delete Bacillus according to claim 1 ( Bacillus velezensis ) culture of KRICT934 (Accession No .: KCTC 11774BP) strain. The Bacillus Belle Zen sheath (Bacillus velezensis) KRICT934 according to claim 1 (Accession No: KCTC 11774BP) strain or a Bacillus Belle Zen system according to 3 wherein (Bacillus velezensis) KRICT934 (Accession No: KCTC 11774BP) containing a culture of the strain Nitrogen mineralization promoting activity of organic matter and antibacterial against one or more causative bacteria selected from rice blast, rice leaf blight, tomato ash mold, tomato blight, wheat rust disease, barley powdery mildew, pepper anthrax, radish rot and pepper blight Microbial agents having simultaneously activity. delete Rice blast, rice leaf blight, tomato ash fungus, tomato blight, wheat red rust, barley flour, pepper anthracnose, radish wilted and pepper A control method of at least one plant disease selected from late blight. delete

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