WO2014175496A1 - Souche de bactéries bacillus methylotrophicus yc7077 endophytiques végétales, biopesticide multifonctionnel l'utilisant et mise au point d'un fertilisant microbien - Google Patents

Souche de bactéries bacillus methylotrophicus yc7077 endophytiques végétales, biopesticide multifonctionnel l'utilisant et mise au point d'un fertilisant microbien Download PDF

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WO2014175496A1
WO2014175496A1 PCT/KR2013/004012 KR2013004012W WO2014175496A1 WO 2014175496 A1 WO2014175496 A1 WO 2014175496A1 KR 2013004012 W KR2013004012 W KR 2013004012W WO 2014175496 A1 WO2014175496 A1 WO 2014175496A1
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strain
plant
rice
growth
bacteria
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정영륜
호세인모하마드토파잘
정유진
김근곤
이정은
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주식회사 제일그린산업
경상대학교산학협력단
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    • C—CHEMISTRY; METALLURGY
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus

Definitions

  • the present invention relates to a plant endogenous microbial strain and a microbial agent containing the same, and more particularly to a Bacillus methylotrophicus new strain and a microbial agent containing the same.
  • the present invention is derived from a study conducted as part of the 'Next Generation Biological Pesticide Development Project' supported by the Ministry of Agriculture, Forestry and Fisheries in 2010.
  • the microbial products developed in this way are selected from microorganisms having a specific function in the environment, formulated after mass cultivation, and treated with foliar spray or granulated soil to control pests or used as microbial fertilizers (see Non-Patent Document 1).
  • Microorganisms used here are widely distributed in the natural environment such as soil, plant roots, and the ocean, and are widely used in environmentally friendly agriculture because they are diverse and secrete various metabolites.
  • There are three ways to use microorganisms including using microorganisms themselves, using metabolites produced by microbial fermentation, and using microbial metabolites as leading compounds for synthesizing new pesticides.
  • antagonistic microorganisms capable of inhibiting plant pathogens, plant growth-promoting myobacterial bacteria (PGPR) and plant endophytic microorganisms, which promote plant growth, are in most cases, especially plant disease resistance by these microorganisms.
  • PGPR plant growth-promoting myobacterial bacteria
  • plant endophytic microorganisms which promote plant growth
  • Endogenous bacteria are defined as bacteria that live in living healthy plant tissues and give various benefits without causing substantial harm to plants.
  • plant endogenous bacteria are present in the spaces between cells or between cells, inducing direct and indirect resistance to plant pests and stress, and are known to have a great influence on plant growth.
  • endogenous bacteria are nitrogen fixation, solubilization of phosphoric acid, production of siderophore, production of active hormones, production of antimicrobial substances or disease resistance of plants to prevent disease infection and promote plant growth. It is considered (refer nonpatent literature 1-4).
  • Such endogenous bacteria are friendly to the host plant and can be symbiotic to the tissues of the host plant without being recognized as pathogens (see Non-Patent Document 5).
  • Some endogenous bacteria can use one or more mechanisms simultaneously to promote plant growth and control disease, and different mechanisms may be applied at different times during the lifetime of the plant.
  • Plant genes may be altered by the presence of bacteria, and these altered genes may provide clues to the effects of endogenous bacteria in plants (see Non-Patent Document 6). Plant endogenous bacteria multiply in cells, usually with a high overall density at the root and at the beginning of the stem, and gradually decreasing toward the upper part of the plant such as stems and leaves, and in the case of corn leaves, the density is log10 3 to log10 7 cfu / g.
  • Bacillus Bacillus
  • Pseudomonas Pseudomonas
  • Enterobacter Enterobacter
  • Agrobacterium Agrobacterium
  • Rice is one of the top five crops in the world, causing a lot of production losses due to various diseases. Among them, damage caused by hospital fungi, such as blast and leaf blight, causes fatal losses in crop production, and has been effectively controlled by breeding resistant varieties and developing chemical fungicides. However, in addition to the kidney disease and bacterial diseases such as alder disease and leaf blight, which are mainly caused by seed transmission occurs a lot depending on the region, causing a lot of damage in recent years have been difficult to control. These pathogens overwinter in paddy soils and surrounding weed tissues, and especially in warm and humid conditions, rice is vulnerable to germs, so it is difficult to control once the onset begins. The control of these pathogens has been mainly dependent on chemical fungicides, but most of the use of fungicides has a problem of environmental pollution or human toxicity, increasing the interest in environmentally friendly biological pesticides that can replace them.
  • Bacillus Bacillus subtilis
  • Bacillus Fu milreoseu Bacillus pumilus
  • Bacillus amyl Lowry Quebec Patience B.
  • amyloliquefaciens is, and the strains most antimicrobial material used in the product It is known to have a mechanism of directly inhibiting germs or to induce host disease resistance by secreting cyclic peptides such as initurin and surfactin.
  • Bacillus sp. Forms endospores that can survive in poor natural environments for long periods of time, and because of their good antibacterial properties, many researchers have published their findings for the biological control of plant diseases. See Patent Document 10).
  • Several studies suggest a trophy for Syracuse (Bacillus methylotrophicus) recent new addition to the endogenous bacteria Bacillus methylation kind mentioned above have been reported. This strain was first identified in Korea in 2010 and identified as having the ability to promote plant growth (see Nonpatent Literature 1).
  • Non-Patent Documents 11 to 13 Have been reported (see Non-Patent Documents 11 to 13).
  • the strain had no effect on the induction of disease resistance of host plants or inhibition of rice bacterial disease in addition to the antibacterial activity against phytopathogens.
  • Bacillus sp. Only a few species are actually commercialized and used effectively in controlling plant diseases. This is considered to be due to the relatively low control effect because it was based only on the mechanism of inhibiting pathogens when separating antagonistic bacteria. Therefore, in order to take advantage of the characteristics of this strain for more effective control, it is necessary to search and develop a new multifunctional microorganism having both antimicrobial activity, host plant disease resistance inducing ability and growth promoting effect.
  • Non-Patent Document 1 Madhaiyan, M., Poongguzhali, S., Kwon, SW, and Sa, TM (2010). Bacillus methylotrophicus sp. nov., a methanol-utilizing, plant-growth-promoting bacterium isolated from rice rhizosphere soil. Int J Syst Evol Microbiol 60: 2490-2495.
  • Non-Patent Document 2 Chert, C., Bauske, EM, Musson, G., Rodriguez-Kabana, R., and Kloepper, JW (1995). Biological control of Fusarium wilt of cotton by use of endophytic bacteria. Biol Cont 5: 10-16.
  • Non-Patent Document 3 Costa, JM, & Loper, JE (1994). Characterization of siderophore production by the biological control agent Enterobacter cloacae . Mol. Plant-Microbe Interact. 7: 440-448.
  • Non-Patent Document 4 Wakelin, SA, Warren, RA, Harvey, PR, Ryder, MH (2004). Phosphate solubilization by Penicillium spp. closely associated with wheat roots. Biology and Fertility of Soils 40: 36-43.
  • Non-Patent Document 5 Rosenblueth, M., & Martinez-Romero, E. (2006). Bacterial endophytes and their interactions with hosts. Molecular Plant-Microbe Interactions 19: 827-837.
  • Non-Patent Document 6 de Matos Nogueira, E., Vinagre, F., Masuda, HP, Vargas, C., de P, VLM., Da Silva, FR, dos Santos, RV, Baldani, JI, Gomes Ferreira, PC , and Hemerley. (2001). Expression of sugarcane genes induced by inoculation with Gluconacetobacter diazotrophicus and Herbaspirillum rubrisubalbicans. Genet. Mol. Biol. 24: 199-206.
  • Non-Patent Document 7 Bibi, F., Yasir, M., Song, G. C., Lee, S. Y., and Chung, Y. R. (2012). Diversity and characterization of endophytic bacteria associated with tidal flat plants and their antagonistic effects on oomycetous plant pathogens. Plant pathol j 28: 20-31.
  • Non-Patent Document 8 Cao, C., Park, S., and McSpadden, G. (2010). Biopesticide controls of plant diseases: resources and products for organic farmenrs in Ohio. Fact Sheet, Agricultural and Natural Resources. SAG-18-10 The Ohio State Univeristy.
  • Non-Patent Document 9 Lee, S. K., Sohn, H. B., Kim, G. G., and Chung, Y. R. (2006). Enhancement of biological control of Botrytis cinerea on cucumber by foliar sprays and bedpotting mixes of Trichoderma harzianum YC459 and its application on tomat in the greenhouse. Plant Pathol J 22: 283-288.
  • Non-Patent Document 10 Park, Kyung-Suk. (2011). Development Status of Microbial Pesticides and the Importance of Bacillus Microorganisms. Industrial Chemistry Prospect 14 (4): 1-11.
  • Non-Patent Document 11 Kim, BK, Chung, JH, Kim, SY, Jeong, H., Kang, SG, Kwon, SK, Lee, CH, Song, JY, Yu, DS, Ryu, CM, and Kim, JF (2012). Genome sequence of the leaf-colonizing bacterium Bacillus sp strain 5B6, isolated from a cherry tree. J Bacteriol 194: 3758-3759.
  • Non-Patent Document 12 Ma, L., Cao, YH, Cheng, MH, Huang, Y., Mo, MH, Wang, Y., Yang, JZ, and Yang, FX (2013). Phylogenetic diversity of bacterial endophytes of Panax notoginseng with antagonistic characteristics towards pathogens of root-rot disease complex. Ant van Leeuwen 103: 299-312.
  • Non-Patent Document 13 Yan, X., He, L., Song, G., and Wang, R. (2011). Antagonistic bioactivity of endophytic strains isolated from Salvia mitiorrhiza . Afr J Biotech 10: 15117-15122.
  • the present invention can suppress the growth of plant pathogenic fungi and bacteria in a way that can compensate for the above disadvantages, and at the same time act specifically on rice, a host plant, to induce disease resistance and promote plant growth.
  • the purpose of the present invention is to develop and provide a new type of biopesticide having a microbial fertilizer efficacy by separating, mass culturing and formulating a multifunctional plant endogenous bacterium.
  • the present invention provides a Bacillus methylotrophicus YC7007 (Accession Number: KCCM11275P) strain having a plant disease resistance induction efficacy.
  • the plant disease provides Bacillus methylotrophicus YC7007 (Accession Number: KCCM11275P) strain, characterized in that at least one selected from the group consisting of rice bran blight, rice bran blight and rice stingray disease.
  • the strain provides Bacillus methylotrophicus YC7007 (Accession No .: KCCM11275P) strain, characterized in that it further has a phytopathogen inhibitory effect and plant growth promoting effect.
  • the plant growth promoting effect provides a plant growth promoting effect on rice Bacillus methylotrophicus ( Bacillus methylotrophicus ) YC7007 (Accession Number: KCCM11275P) provides a strain.
  • the present invention provides a microbial preparation for fertilizers and bio-pesticides containing the strain or its culture as an active ingredient.
  • the present invention it is possible to simultaneously inhibit the growth of important phytopathogenic fungi and bacteria of crops, and the multifunctional bacterium Bacillus methylotrophicus YC7007 strain having both the disease resistance induction and the growth promoting effect of host plants It is a product that contains multifunctional bacteria that not only directly inhibit phytopathogenic bacteria by secreting antibiotics by using antibiotics, but also acts specifically on rice to promote growth and induce systemic disease resistance. It is possible to provide an excellent microbial agent that can simultaneously serve as a pesticide and microbial fertilizer.
  • 1 is a nucleotide sequence of 16S rRNA gene of YC7007 strain
  • Figure 2 is a phylogenetic tree made by 16S rRNA gene sequence analysis of YC7007 strain
  • Figure 3 is a photograph showing the result of the gene comparison analysis compared to the standard strain using the gene analysis (BOX-PCR) of the YC7007 strain,
  • Figure 4 is a comparison photo for each treatment group illustrating the effect of rice blight control
  • Figure 5 is a comparison photo for each treatment group to explain the effect of controlling rice leaf blight
  • FIG. 6 is a graph (a) and photograph (b) showing the antimicrobial effect of each culture period of the culture filtrate of YC7007 strain;
  • Figure 7 is a graph showing the antimicrobial effect of the YC7007 strain culture filtrate according to the temperature treatment.
  • Example 1 Isolation and Identification of YC7007 Strains
  • the YC7007 strain was isolated from the root internal tissue of Cyperus sp. Growing in paddy fields of Gyeongsang National University.
  • the surface of the root cut pieces was immersed in 1% sodium hypochlorite (NaOCl) solution for 10 minutes to sterilize the surface.
  • NaOCl sodium hypochlorite
  • These pieces were placed on 1 / 10TSA medium (Tryptic Soy Broth 3g, agar 16g / 1 liter of distilled water), and cultured for about 2 to 3 days while observing the growth of bacteria on the surface to confirm surface sterilization.
  • 1.0g of the root pieces of which the surface sterilization of the pieces were confirmed were taken, and 9.0ml of sterilized distilled water was put in a high-pressure sterilizer and ground into sterilized bowls and mortars.
  • the isolated YC7007 strain was Gram-positive, non-motile, cells were rod-shaped (0.6 ⁇ m wide, 1.8-2.6 ⁇ m long), formed endospores, and were unable to grow under anaerobic conditions.
  • the results of examining the physiological and biochemical characteristics of this strain and similar species are shown in Table 1, which was identified as Bacillus methylotrophicus .
  • strains 1 and 2 are investigation results according to the analysis method
  • strains 3 and 4 are non-patent literature 13
  • Non-Patent Document 14 Sudpavapol, P., Tongyonk, L., Tanasupawat, S., Chokesajjawatee, N., Luxananil, P., and Visessanguan, W. (2011) .Bacillus siamensia sp.nov., Isolated from salted crab ( poo-khem ) in Thailand.Int J Syst Evol Microbio
  • the physiological and biochemical characteristics of the YC7007 strain were compared with other similar Bacillus species, and were grown in 10% salt solution, at 10 ° C and 50 ° C, ortho-nitro-phenyl- ⁇ - Acid production from ortho-nitro-phenyl- ⁇ -D-galactopyranoside, sodium citrate, sodium pyruvate, esterase lipase from API zym kit test (C8)), ⁇ -glucosidase, ⁇ -glucosidase reactions, and important phenotypic characteristics such as whether D-xylose is used in API 50CH kit tests It can be seen that the YC7007 strain is different from the related species.
  • Figure 2 shows the phylogenetic tree made by 16S rRNA gene sequence analysis of the YC7007 strain.
  • the intersection number in FIG. 2 represents the bootstrap value resulting from 1000 iterations.
  • Figure 3 shows the results of gene comparison analysis compared to the standard strain using the gene analysis (BOX-PCR) of the YC7007 strain.
  • Polymerase for gene analysis (BOX-PCR) was used as Platinum Taq DNA polymerase High Fidelity (Invitrogen), and primer was used as BOXAR1 (5'-CATCGGCAAGGCGACGCTGACG-3 ').
  • PCR conditions include initial denaturation at 95 ° C. for 7 minutes, denaturation, annealing and extension at 90 ° C. for 30 seconds, 40 ° C. for 1 minute, Finally, the final extension was amplified by reacting at 72 ° C for 10 minutes for 3 minutes at 72 ° C.
  • M represents a 1 kb marker
  • 1 represents a YC7007 strain
  • 2 represents a YC7010 strain
  • 3 represents a Bacillus methylotrophicus KACC13105 standard strain.
  • the gene analysis (BOX-PCR) was performed for a more detailed comparison with the same standard strain with the YC7007 strain, and the YC7007 strain was found to be different from the standard strains.
  • the microorganism of the present invention was named Bacillus methylotrophicus YC7007 strain and received the accession number KCCM11275P from the Korean Culture Center of Microorganisms (KCCM) on April 18, 2012.
  • Example 2 In Vitro Inhibitory Effect Assay for Pathogenic Fungi and Bacteria of YC7007 Strains
  • Antimicrobial activity of endogenous bacteria is an important plant pathogenic fungi YC7007 10 kinds (Gibberella fujikuroi (Rice tall bottles), Colletotrichum acutatum (Anthrax), Fusarium oxysporum f.sp.
  • cucumerinum cucumber wilt
  • Sclerotinia sclerotiorum gyunhaekbyeong
  • Pythium ultimum damping-off
  • Bipolaris oryzae rice flakes
  • Magnaporthe grisea rice fever
  • Botrytis cinerea ash fungus
  • Botryosphaeria dothidea leaf rot
  • Rhizoctonia solani rice leaf blight
  • four pathogenic bacteria Burkholderia glumae
  • Xanthomonas oryzae pv cucumerinum (cucumber wilt)
  • Sclerotinia sclerotiorum gyunhaekbyeong
  • Pythium ultimum damping-off
  • Bipolaris oryzae rice flakes
  • Magnaporthe grisea rice fever
  • Botrytis cinerea ash fungus
  • Botryosphaeria dothidea leaf rot
  • the pathogenic fungi and YC7007 strains inhibit growth by using a paper disc method in 1 / 5PDA and 1 / 10TSA of growth medium (16g agar / 1 liter of distilled water) including potato agar sugar medium (PDB, Difco) Was investigated.
  • a paper disk (5 mm in diameter) was placed 1 cm from the edge of the incubator, 100 ⁇ l of the strain culture was soaked into the paper disk, and then placed in the middle of the pathogen fungal mycelium disk (6 mm) grown for 4 days in PDA medium. According to the incubation at 24 ⁇ 28 °C 4-6 days.
  • Bacterial inhibitory effect of culture filtrate produced by strain YC7007 was shaken (120rpm) at 28 °C for 72 hours in gastric culture medium except agar, and the culture solution was filtered by millipore filter after centrifugation. The filtrate was used.
  • the inhibitory effect after the treatment for 10 minutes in hot water maintained at each treatment temperature was investigated.
  • Table 2 and Table 3 show the results of the antimicrobial activity of the main phytopathogenic fungi and major phytopathogenic bacteria of the YC7007 strain.
  • the antimicrobial activity of the phytopathogenic fungi of the endogenous bacteria YC7007 strain is slightly different depending on the medium, but it can be seen that exhibited 3 to 10 mm mycelial growth inhibitory effect.
  • the culture medium was 6 ⁇ 10mm, the culture filtrate showed a growth inhibitory effect of 3 ⁇ 8mm.
  • the control effect of the seven major plant diseases of the YC7007 strain was investigated as follows.
  • the control efficacy test was performed by the Korea Research Institute of Chemical Research.
  • the plant diseases were rice blast, rice leaf blight, tomato ash fungus, tomato blight ( Phytophthora infestans ), wheat rust disease ( Puccinia recondita ), barley Powdery mildew (pathogen:.. Blumeria graminis f sp hordei ) and pepper anthracnose: It was (pathogen Colletotrichum coccodes).
  • Rice Blast (RCB) Magnafoss Greek, a pathogen M. grisea ) Inoculated KJ201 strain into rice bran agar medium and incubated for 2 weeks in a 25 °C incubator to harvest spores formed with sterile distilled water (3x10). 5 spores / ml) was prepared. It was sprayed sufficiently to flow down to the drug-treated Nakdong rice (3-4 leaves of the main leaf). Inoculated rice was incubated for 24 hours in a dark state in a wet room, followed by 4 days in a constant temperature and humidity room with a relative humidity of 80% or higher and a temperature of 26 ° C.
  • Rice Blister Blight (RSB) is sterilized by putting an appropriate amount of bran into a 1l bottle, R. solani AG1 After the inoculation of the strain was incubated for 7 days in a 25 °C incubator. The bottle inoculation was inoculated with a finely pulverized mycelium mass and inoculated evenly in the pot where Nakdong rice of the 4-5 leaves treated with the drug was incubated and incubated for 7 days in a wet room (25 ° C.). The lesion area ratio formed in the sheath was investigated.
  • Tomato gray mold disease was inoculated with the pathogen, Botrytis cinerea ( B. cinerea ) in potato agar medium and cultured in a 25 °C thermostat was used as inoculum.
  • Botrytis cinerea B. cinerea
  • the bottle was inoculated with spores harvested using a hemocytometer to make a spore concentration of 3x10 5 spores / ml and sprayed with tomato seedlings (2 to 3 leaves) treated with the drug.
  • Inoculated tomato seedlings were placed in a 20 ° C. wet room (relative humidity of 95% or more) to induce onset for 3 days, and then the lesion area ratio was examined.
  • Tomato late blight was inoculated with the pathogen P. infestans strain in oatmeal medium and cultured in an incubator at 20 °C to form a jujube sac.
  • Pathogen inoculation was harvested by adding sterile distilled water and spore suspension having a spore concentration of 3 ⁇ 10 4 spore sacs / ml. They were put in a refrigerator and subjected to a low temperature treatment so that the yuzu was spilled to prepare a yuzu suspension. This was sprayed onto the drug-treated tomato seedlings (two to three leaves). Tomato seedlings inoculated with the pathogens were treated in a 20-degree wethouse for 2 days and inoculated in a constant temperature and humidity room.
  • Wheat red rust is a pathogen, P. recondita , is a live parasite. Therefore, the spores formed in wheat seedlings were used as inoculum while the plants were passaged directly to plants.
  • five seed grains (variety: silver onion) were sown in disposable pots (4.5cm in diameter) and treated with a single leaf seedling seedling grown in a greenhouse for 8 days, and then inoculated (spore 0.11g / 1) was sprayed. Inoculated wheat seedlings were incubated at 20 ° C. for 1 day and then transferred to a constant temperature and humidity room at 20 ° C. with a relative humidity of 70% to induce onset.
  • Barley powdery mildew (BPM) is a pathogen, B. graminis f. Sp.hordei , is a live parasite, and was used as an inoculum in barley seedlings as a barley seedling in the laboratory. Pharmacokinetic investigation was carried out by dispensing five barley seeds (variety: copper barley) in a disposable pot (diameter: 4.5 cm), spraying the medicinal plant on the first leaf seedlings grown for 8 days in a greenhouse, air-dried in the greenhouse, Powdery mildew spores were inoculated. Inoculated barley seedlings were placed in a constant temperature and humidity room at 20-23 ° C and a relative humidity of about 60% for 7 days, and then the area ratio was investigated.
  • Pepper anthrax was inoculated with the pathogen C. coccodes in oatmeal medium and incubated at 25 ° C. for 10 days to harvest the formed spores and adjust the spore concentration to 2 ⁇ 10 5 spores per ml.
  • the spore suspension was prepared.
  • the spore suspension prepared in the pepper seedlings (3-4 leaves) treated with the drug was sprayed and put in a wet bed (25 °C) and treated for 2 days and then developed in a constant temperature and humidity room (25 °C, 75% RH). Three days after the inoculation, the lesion area formed on the pepper leaves was examined.
  • the control value was calculated by calculating according to the following formula 1 from the disease area ratio obtained from the bottle irradiation.
  • Table 4 shows the results of the control effect on the major plant diseases of the YC7007 strain.
  • Example 4 Rice erosion control assay by inducing disease resistance of YC7007 strain
  • the pathogen suspension was incubated in R2A medium for 24 hours and then centrifuged to suspend bacterial cells in a 10 mM MgSO 4 solution and to adjust the concentration appropriately.
  • the degree of lesion necrosis was examined. The incidence was divided into 0-3 (0: no symptom, 1: small necrotic spot, 2: several necrotic spots, large gallbladder, 3: total necrosis), and the control value was calculated by calculating the degree of disease inhibition on the control. .
  • Benzothiadiazole (BTH) a 1 mM solution, was used as a control agent for inducing rice disease resistance.
  • Table 5 shows the results of investigation of the control effect of rice bran blight by the soil treatment of the YC7007 strain
  • Figure 4 shows a comparison picture for each treatment to explain the effect of rice blight blight control.
  • the strain treatment group had a leaf path length of 2.0 mm and an incidence of 1.1.
  • the length of disease was 17.4mm and the incidence was much less than that of 2.4.
  • the control value was 54%, which was equivalent to 57% of BTH, the control drug.
  • necrosis degree was examined. The incidence is divided into 0-9 (0: no symptom, 1: small necrotic spot, 2-4: several necrotic spots combined, medium reed, 5-8: several necrotic spots combined, large reed, 9: total necrosis)
  • the control value was calculated by calculating the degree of disease inhibition for the control.
  • Table 6 shows the results of the control of rice leaf blight control effect by soil treatment of YC7007 strain, and in FIG. 5 shows a comparison picture for each treatment group to explain the effect of control of rice leaf blight.
  • Rice seedlings (Dongjin 1) grown in plant growth conditions (28 ⁇ 30 °C, relative humidity 80% or more) for 2 weeks are transferred to sterile or non-sterile rice cultivation soil and planted immediately after suspension (10 5 ⁇ 10 7 cfu / ml) 15 ml) was aliquoted into topsoil (150 g). After 3 days of treatment, G. fujikuroi ( 5 ⁇ 10 5 cfu / g) was inoculated at 1.5 g / pot in 10 seedlings per repetition in three repetitions. The incidence of the disease was examined after placing it on growth for 10-30 days after inoculation.
  • the incidence was divided into 0-5 (0: no symptom, 1: leaf slightly yellowing and stem dwarfism, 2 ⁇ 3: leaf yellowing and stem tallening, 5: total necrosis), and the control group calculated the degree of disease inhibition for the control. Calculated by
  • Table 7 shows the results of investigating the control effect of rice stinging disease by soil treatment of YC7007 strain.
  • Example 7 Growth Promotion Effect of Rice by YC7007 Strain Treatment
  • Table 8 shows the results of investigating rice growth promoting efficacy of the YC7007 strain.
  • the YC7007 strains were treated in rice growing soils and grown in pots to investigate the growth promoting effect of each growing season.
  • the YC7007 strains were found to be 11.7 cm and 2.7 cm long in both seedlings. It was much longer than 19.3cm and 7.7cm, and stem length was 27.4% more and even 52% more numbered.
  • the YC7007 strain treatment group also showed a 10.8% increase in stem length and 32% more crushed number in the extracting season, confirming the effect of promoting the growth of rice by the YC7007 strain treatment.
  • the production of antimicrobial substances was investigated in 1 / 10TSB liquid medium.
  • the degree of inhibitory effect of the antimicrobial substance was confirmed by measuring the fungal hyphae or bacterial cell growth inhibition distance formed around the paper disk by the above-described disk diffusion method.
  • the YC7007 strain was incubated at 28 ° C. for 72 hours in a liquid medium (180rpm), centrifuged at 9000 g for 10 minutes, and the culture filtrate was filtered with a Millipore filter (0.2 ⁇ m), and then the antimicrobial activity was measured for each culture time.
  • FIG. 6 is a graph (a) and photograph (b) showing the antimicrobial effect of each culture period of the YC7007 strain culture filtrate
  • Figure 7 is a graph showing the antimicrobial effect of the temperature treatment of the YC7007 strain culture filtrate.
  • Bacteria culture obtained by culturing the strain in a large fermenter (1 ton or more) or cells obtained after centrifugation were mixed at 1: 100 with clay minerals such as kaolin, bentonite, and peat and dried at low temperature. After uniformly pulverized to prepare a solid in the form of a powder. At the same time, the bacterial cultures were also mixed with the clay minerals 1: 100 to prepare a liquid suspension.
  • SEQ ID NO: 1 is a nucleotide sequence of 16S rRNA gene of another YC7007 strain.

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Abstract

L'invention concerne une nouvelle souche utilisée pour un nouveau type de biopesticide, présentant un effet de fertilisation microbien, par séparation, culture de la masse et préparation de bactéries endophytiques végétales multifonctionnelles présentant tous les effets d'une prévention considérable de la croissance de champignons et de bactéries pathogènes pour les plantes et étant appliquée simultanément, plus particulièrement sur le riz, qui est la plante hôte, induisant ainsi une résistance aux maladies et favorisant ainsi la croissance des plantes. La présente invention concerne un Bacillus methylotrophicus YC 7077 (numéro d'accès : KCCM 11275P) présentant tous les effets d'une prévention de la croissance de champignons et de bactéries pathogènes pour les plantes, favorisant la croissance des plantes et induisant une résistance aux maladies des plantes dans une plante hôte.
PCT/KR2013/004012 2013-04-25 2013-05-08 Souche de bactéries bacillus methylotrophicus yc7077 endophytiques végétales, biopesticide multifonctionnel l'utilisant et mise au point d'un fertilisant microbien WO2014175496A1 (fr)

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KR10-2013-0046313 2013-04-25

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CN105557756A (zh) * 2016-02-05 2016-05-11 中国农业科学院植物保护研究所 利用甲基营养型芽孢杆菌nkg-1实施的植物抑菌方法
CN107267412A (zh) * 2017-05-24 2017-10-20 浙江大学 甲基营养型芽孢杆菌及其应用
WO2018021797A1 (fr) * 2016-07-28 2018-02-01 전남대학교산학협력단 Souche dr-08 de bacillus methylotrophicus produisant un composé volatil naturel et présentant une activité antibactérienne et utilisation correspondante
CN108085284A (zh) * 2018-01-24 2018-05-29 大连理工大学 甲基营养型芽孢杆菌及包含该微生物的菌剂制备方法与应用
US10856551B2 (en) * 2014-07-31 2020-12-08 Universidad De Granada Use of Bacillus methylotrophicus as a stimulant of plant growth and biological control means, and isolates of said species
CN114621946A (zh) * 2022-04-07 2022-06-14 施可丰化工股份有限公司 一种防治大豆根腐病的生防菌剂及其制备方法
CN115975847A (zh) * 2022-08-08 2023-04-18 兰州交通大学 一种用于防治当归根腐病的特基拉芽孢杆菌及其应用
CN117187129A (zh) * 2023-09-05 2023-12-08 北京市水产技术推广站(北京市鱼病防治站) 一株产ddp-iv抑制剂的阴沟肠杆菌及其应用

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KR101869790B1 (ko) * 2016-11-10 2018-06-21 대한민국 신규 미생물 바실러스 메틸로트로피쿠스 cc112 또는 이를 함유하는 미생물 제제
KR102656677B1 (ko) 2021-11-11 2024-04-12 (주)에코비즈넷 식물병 방제 효능이 우수한 바실러스 서브틸리스 bc-6 균주 및 이의 용도
KR102656678B1 (ko) 2021-11-11 2024-04-12 (주)에코비즈넷 식물 생장 촉진능 및 식물병 방제 효능이 있는 바실러스 벨레젠시스 jc-9 및 이의 용도

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US10856551B2 (en) * 2014-07-31 2020-12-08 Universidad De Granada Use of Bacillus methylotrophicus as a stimulant of plant growth and biological control means, and isolates of said species
CN105557756A (zh) * 2016-02-05 2016-05-11 中国农业科学院植物保护研究所 利用甲基营养型芽孢杆菌nkg-1实施的植物抑菌方法
US11528912B2 (en) 2016-07-28 2022-12-20 Global Agro Co., Ltd. Bacillus methylotrophicus strain DR-08 producing natural volatile compound and having antibacterial activity, and use thereof
WO2018021797A1 (fr) * 2016-07-28 2018-02-01 전남대학교산학협력단 Souche dr-08 de bacillus methylotrophicus produisant un composé volatil naturel et présentant une activité antibactérienne et utilisation correspondante
CN107267412A (zh) * 2017-05-24 2017-10-20 浙江大学 甲基营养型芽孢杆菌及其应用
CN108085284A (zh) * 2018-01-24 2018-05-29 大连理工大学 甲基营养型芽孢杆菌及包含该微生物的菌剂制备方法与应用
CN108085284B (zh) * 2018-01-24 2021-03-30 大连理工大学 甲基营养型芽孢杆菌及包含该微生物的菌剂制备方法与应用
CN114621946A (zh) * 2022-04-07 2022-06-14 施可丰化工股份有限公司 一种防治大豆根腐病的生防菌剂及其制备方法
CN114621946B (zh) * 2022-04-07 2023-07-04 施可丰化工股份有限公司 一种防治大豆根腐病的生防菌剂及其制备方法
CN115975847A (zh) * 2022-08-08 2023-04-18 兰州交通大学 一种用于防治当归根腐病的特基拉芽孢杆菌及其应用
CN115975847B (zh) * 2022-08-08 2023-09-26 兰州交通大学 一种用于防治当归根腐病的特基拉芽孢杆菌及其应用
CN117187129A (zh) * 2023-09-05 2023-12-08 北京市水产技术推广站(北京市鱼病防治站) 一株产ddp-iv抑制剂的阴沟肠杆菌及其应用
CN117187129B (zh) * 2023-09-05 2024-03-12 北京市水产技术推广站(北京市鱼病防治站) 一株产ddp-iv抑制剂的阴沟肠杆菌及其应用

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