KR20140127670A - Endophytic bacteria Bacillus Methylotrophicus YC7007 and development of a multifunctional biopesticide and microbial fertilizer using same - Google Patents

Abstract

Disclosed is a novel strain which can inhibit growth and development of plant-pathogenic fungus and virus extensively, specifically acts on rice which is a host plant, separates and mass-cultures multifunctional plant endogenous bacteria having effects of inducing disease resistance and promoting growth and development of plants, and manufactures a medicine, thereby being used as a novel biopesticide having microorganism fertilizer effect. Provided is a Bacillusmethylotrophicus YC7007 (accession number: KCCM11275P) strain having effects of inhibiting growth and development of plant-pathogenic fungus and virus, promoting growth and development of plants, and inducing disease resistance of a host plant.

Description

[0001] Description [0002] The present invention relates to a bacterial endogenous bacterium Bacillus methylotrophicus YC7007 strain, and a multifunctional biocidal pesticide and a microbial fertilizer using the same.

The invention trophy kusu (Bacillus include Bacillus methyl relates to a preparation containing microbial plant endogenous microbial strains and this, and more specifically methylotrophicus ) and a microorganism preparation containing the same.

The present invention was derived from research carried out as part of the 'next generation biopesticide development project' supported by the Ministry of Food, Agriculture, Forestry and Fisheries in 2010.

[Task No.: 808015-3-2, Title of the Project: Development of next-generation microbial materials for plant disease control using antagonistic microorganisms (2010-2013)]

Globally, over the past several decades, we have been using chemical fertilizers and chemical pesticides for disease, pest and weed control each year to increase the yield of crops such as grains, vegetables, and fruit trees. Such continuous use of chemical fertilizers and pesticides has continuously raised problems such as soil acidification, loss of power, environmental pollution and ecosystem destruction, lethal toxicity, pest resistance and resistance to pesticides in weeds.

In order to solve these problems at home and abroad, efforts are being made to reduce the use of chemical fertilizers and pesticides. In particular, the Korean government has enacted the 3rd Environmentally Friendly Farming Act in 1999, The goal of the plan is to reduce chemical fertilizer and pesticide use by about 15% At the same time, the public is increasingly demanding safer food due to the improvement of living standards. Environment-friendly agriculture such as organic farming and natural farming methods that are aiming to preserve agricultural production environment and natural ecosystem are rapidly expanding. And studies on the development of fertilizers and biopesticides using microorganisms that can replace pesticides have been rapidly increasing. The microbial products thus developed are selected from environmentally selected microorganisms, cultivated in large quantities, formulated, and subjected to soil treatment by spraying with leaves or by spraying with a granule, thereby controlling pests or being used as microbial fertilizers (see Non-Patent Document 1).

The microorganisms used here are widely distributed in natural environments such as soil, plant rhizosphere, and ocean, and they are used effectively in environmentally friendly agriculture because they are various kinds and secrete various kinds of metabolites. The use of microorganisms can be roughly classified into three categories: a method using microorganisms themselves, a method using metabolites produced by microbial fermentation, and a method using microbial metabolites as a lead compound for the synthesis of new pesticides. Among them, antagonistic microorganisms capable of inhibiting plant pathogens, PGPR promoting plant growth and PGPR and endophytic microorganisms are most directly used, and in particular, plant disease resistance caused by these microorganisms Much research is underway.

In recent 10 years, researches on endophytic bacteria have been actively conducted among various microorganisms in domestic and foreign countries. Endophytic bacteria are defined as bacteria which live in living healthy plant tissues and give various advantages without harming plants . In general, endophytic bacterium is known to exist in spaces or cells between plant cells and directly or indirectly induce resistance to plant pests, stress, and also have a great influence on plant growth. More specifically, the endogenous bacteria prevent the disease infection and promote plant growth by inducing nitrogen fixation, solubilization of phosphoric acid, siderophore production, active hormone production, antimicrobial substance production or plant disease resistance (See Non-Patent Documents 1 to 4). Such endophytic bacteria are friendly to the host plants and can not be recognized as pathogens and can symbiosis with the host plant tissues (see Non-Patent Document 5). Certain endogenous bacterium may utilize one or more mechanisms simultaneously to promote plant growth and disease control effects, and different mechanisms may be applied at different times during the life of the plant. Plant genes may be altered by the presence of bacteria, and such altered genes may provide clues to the effects of endogenous bacteria in plants (see Non-Patent Document 6). Plant endophytic bacteria proliferate intracellularly, and generally have a high density at the beginning of roots and stems, and gradually decrease toward the upper part of plants such as stems and leaves. In the case of corn leaves, their density is log10 ³ to log 10 ⁷ cfu / g . The endogenous bacteria isolated to date, Bacillus (Bacillus), Pseudomonas (Pseudomonas), Enterobacter (Enterobacter), Agrobacterium (Agrobacterium), etc., and W in the 50 130 paper is known, all of which are such as tomatoes, potatoes, corn, and rice It promotes the production of crops by suppressing the growth and disease occurrence of economically important crops.

Rice is one of the five major crops in the world and many production losses are caused by various diseases. Among them, damage caused by pathogenic fungi such as blast foliage, sheath blight, and the like is a fatal damage to the crop production. So far, it has been effectively controlled through the development of resistant breeding and chemical fungicide. However, in addition to this, Kidari disease, which is caused by seed transmission, and bacterial disease, such as egg blight and blight of blight, occur in many regions and cause great damage. These pathogens are wintering in paddy soil or surrounding weeds, and especially in warm and humid conditions, rice is vulnerable to germs. Although the control of these pathogens has been mainly dependent on chemical sterilizers, the use of most microbicides is becoming a concern for environmentally friendly biological pesticides that can replace them due to environmental pollution or the risk of lethal toxicity.

Bacterial bacillus ( Bacillus ) and fungus trichoderma ( Trichoderma ) were used to identify the most effective bio-pesticides for the control of plant diseases, which have been developed and sold to date. (Refer to Non-Patent Documents 7 to 10). Bacillus (Bacillus) The kind of Bacillus subtilis (B. subtilis), Bacillus Fu milreoseu (B. pumilus), and Bacillus amyl Lowry Quebec Patience (B. amyloliquefaciens) is, and the strains most antimicrobial material used in the product It is known that it secretes cyclic peptides such as iturin and surfactin to directly inhibit germs or to induce host disease resistance. Bacillus sp. Forms an endospore that can survive for a long time in poor natural environments. Many researchers have published research results for the biological control of plant diseases because of their good antimicrobial activity Patent Document 10). Recently, a new trophy to Syracuse endogenous bacterium Bacillus methylation in addition to the above-mentioned type (Bacillus methylotrophicus ) have been reported. This strain was first isolated in Korea in 2010 and confirmed to be capable of promoting plant growth (see Non-Patent Document 1), followed by isolation from ginseng, tomato and potato rhizome in China, (See Non-Patent Documents 11 to 13). However, in these studies, there is no evidence for the induction of disease resistance or inhibition of bacterial disease in host plants other than the antimicrobial activity against the plant pathogens. Bacillus sp.), there are only a few species that are actually commercialized and effectively used for plant disease control. This may be due to the fact that the control effect is relatively low because only the mechanism of inhibiting pathogens is used as a standard when isolating antagonistic bacteria. Therefore, it is necessary to search for and develop new multifunctional microorganisms having antibacterial activity, inducing disease resistance of host plants and promoting growth for effective control while utilizing the characteristics of this strain.

[Non-Patent Document]

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. deP, 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, GC, Lee, SY, and Chung, YR (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 farms 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: (2011). Development status of microbial pesticides and importance of Bacillus microorganisms. Industrial Chemical Outlook 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, (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 properties 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.

Accordingly, the present invention can broadly inhibit the growth of plant pathogenic fungi and bacteria by supplementing the disadvantages of the above, and at the same time, it specifically acts on the host plant, rice, to induce disease resistance and promote plant growth The present invention aims to develop and provide a new type of pesticide having microbial fertilizer effect by preparing a multifunctional plant endophytic bacterium by isolating and mass-culturing it.

The present invention to solve the aforementioned problems is a trophy kusu (Bacillus of the Bacillus methyl having plant disease resistance induced effects methylotrophicus ) YC7007 (accession number: KCCM11275P).

In addition, a trophy kusu (Bacillus of the Bacillus methyl, characterized in that said plant disease is at least one member selected from the group consisting of rice al blight, rice blight and rice huinip tall bottle methylotrophicus ) YC7007 (accession number: KCCM11275P).

In addition, the strain may be a Bacillus methylotrophicus ( Bacillus < RTI ID = 0.0 > methylotrophicus ) YC7007 (accession number: KCCM11275P).

In addition, the plant growth promoting effect of the present invention is a Bacillus methylotrophicus ( Bacillus sp. methylotrophicus ) YC7007 (accession number: KCCM11275P).

In order to solve the above-mentioned problems, the present invention provides a microorganism preparation for a fertilizer and a biological pesticide containing the strain or a culture solution thereof as an active ingredient.

The present invention also provides a microorganism preparation for a fertilizer and a microbial agent for a pesticide, which contains, as an effective ingredient, a physiologically active substance exhibiting the above-mentioned effect in the above-mentioned strain or a culture solution thereof.

According to the present invention, it is possible to provide a multifunctional bacterium Bacillus ( Bacillus subtilis), which can inhibit important plant pathogenic fungi and bacterial growth of crops at the same time and induce disease resistance and growth promotion of host plants, Methylotrophicus YC7007 is a multifunctional bacterium containing both antimicrobial antibiotics directly inhibiting plant pathogens and rice bacterium that is specific for rice and promotes growth and induces systemic disease resistance. It is possible to provide an excellent microorganism preparation capable of simultaneously performing a role as a biological pesticide and a microorganism fertilizer.

1 shows the nucleotide sequence of 16S rRNA gene of YC7007 strain,
Fig. 2 shows the results of a 16S rRNA gene sequence analysis of strain YC7007,
FIG. 3 is a photograph showing the result of comparative analysis of standard strain versus standard strain using gene analysis (BOX-PCR) of YC7007 strain,
Fig. 4 is a comparative photograph showing the effect of controlling rice blight-preventing disease,
Fig. 5 is a comparative photograph showing the effect of controlling rice blast fungus blight prevention,
FIG. 6 is a graph (a) and a photograph (b) showing the antibacterial effect of the YC7007 strain culture filtrate for each culture period,
7 is a graph showing the antimicrobial effect of the YC7007 culture filtrate by temperature treatment.

Hereinafter, the present invention will be described in detail with reference to examples. Prior to this, terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms, and the inventor should appropriately interpret the concepts of the terms appropriately The present invention should be construed in accordance with the meaning and concept consistent with the technical idea of the present invention. Accordingly, it is to be understood that the constituent features of the embodiments described herein are merely the most preferred embodiments of the present invention, and are not intended to represent all of the inventive concepts of the present invention, so that various equivalents, And the like.

Example  One: YC7007  Isolation and Identification of Strain

(1) Isolation of YC7007 strain

The strain YC7007 was isolated from the root tissue of rice weevil ( Cyperus sp.) Grown in the paddy soil of Gyeongsang National University.

First, in order to isolate endophytic bacteria, the surface of root cut pieces was immersed in 1% hypochlorite (NaOCl) solution for 10 minutes to sterilize the surface. These fragments were placed on a 1 / 10TSA medium (3 g of Tryptic Soy Broth, 16 g of agar / 1 L of distilled water), cultured for 2 to 3 days, and bacterial growth on the surface was observed to confirm sterilization of the surface. 1.0 g of a piece of the surface of which the surface was sterilized was taken and 9.0 ml of sterilized distilled water was put in a high pressure sterilizer, and the mixture was ground with a sterilized flour and mortar. 0.1 ml of this milled solution was added to 0.9 ml of sterilized distilled water, diluted 10-fold ( ^10-3 ), and further diluted 10-fold (10 ^-4 and 10 ^-5 ) sequentially. The diluted solution was divided into three equal portions And then smoothed evenly. The incubator was incubated at 28 ° C for 2 to 3 weeks, and the single colonies which were grown were separated purely and named as YC7007 strain.

(2) Identification of YC7007 strain

In order to identify the isolated YC7007 strain, morphological characteristics, physiological and biochemical characteristics were investigated as follows. The morphological characteristics were examined by scanning electron microscope and the physiological and biochemical characteristics were examined using the analytical method and general analysis method developed by the present inventor Non-patent document 7).

The isolated YC7007 strain was Gram - positive and had no motility, and the cells were rod - shaped (width 0.6 ㎛, length 1.8 ~ 2.6 ㎛) and formed endospores and were unable to grow under anaerobic conditions. The results of investigation of physiological and biochemical characteristics with this strain and similar species are shown in Table 1, and this strain was identified as Bacillus methylotrophicus . The strains No. 1 and No. 2 in Table 1 are the results of the analysis according to the above analysis method and the strains No. 3 and No. 4 are respectively the non-patent document 13 (Gatson, JW, Benz, BF, Chandrasekaran, C., Satomi, M., Venkateswaran, K., and Hart, ME (2006). Bacillus tequilensis sp. Nov. , Isolated from a 2000-year-old Mexican shaft-tomb, is closely related to Bacillus subtilis . Int J Syst Evol Microbiol 56: 1475-1484), non-patent document 14 (Sumpavapol, P., Tongyonk, L., Tanasupawat, S., Chokesajjawatee, N., Luxananil, P., and Visessanguan, Bacillus siamensia sp. nov., isolated from salted crab ( poo-khem ) in Thailand. Int J Syst Evol Microbiol 60: 2364-2370).

* Day

- 1: YC7007

- 2: B. methylotrophicus KACC13105 ^T

- 3: B. tequilensis 10b ^T

- 4: B. siamensis PD-A10 ^T

- +: positive, -: negative, ND: not investigated

As shown in Table 1, the physiological and biochemical characteristics of the YC7007 strain were compared with other similar Bacillus species. As a result, it was found that growth in 10% salt solution, growth at 10 ° C and 50 ° C, Acid production from ortho-nitro-phenyl- beta -D-galactopyranoside, sodium citrate, sodium pyruvate, esterase lipase from API zym kit test, (C8)), α-glucosidase, β-glucosidase reaction and API 50CH kit test using D-xylose The YC7007 strain is different from the related species.

On the other hand, the nucleotide sequence of the 16S rRNA gene of YC7007 strain (1513 bp, see FIG. 1) was determined and the genealogical position was examined by performing homology search with the database of GenBank / EMBL / DDBJ. Fig. 2 shows the phylogeny of 16S rRNA gene sequence analysis of YC7007 strain. In FIG. 2, the intersection number represents the bootstrap value from 1000 repetitions.

FIG. 3 shows the result of comparative analysis of standard strain versus standard strain using gene analysis (BOX-PCR) of strain YC7007. Platinum Taq DNA polymerase High Fidelity (Invitrogen) was used as a polymerase for gene analysis (BOX-PCR) and BOXAR1 (5'-CATCGGCAAGGCGACGCTGACG-3 ') as a primer. The denaturation, annealing, and extension of the initial denaturation at 95 ° C for 7 minutes and 35 times were performed at 90 ° C for 30 seconds, at 40 ° C for 1 minute, 72 ° C for 3 min, and final extension at 72 ° C for 10 min. The amplified PCR product was amplified using 1% LE agarose gel (Seakem). 3, M represents a 1 kb marker, 1 represents a YC7007 strain, 2 represents a YC7010 strain, and 3 represents a Bacillus methylotrophicus KACC13105 standard strain.

As shown in FIG. 3, the genetic analysis (BOX-PCR) was performed for a more detailed comparison with a standard strain having the same scientific name as the strain YC7007. As a result, the strain YC7007 was found to be different from the standard strain.

Accordingly, the microorganism of the present invention is designated as Bacillus methylotrophicus YC7007 strain and received accession number KCCM11275P from the Korean Culture Center of Microorganisms (KCCM) on Apr. 18, 2012.

Example  2: YC7007  For the pathogenic fungi and bacteria of the strain In the incubator  Inhibition efficacy test

The antibacterial activity of YC7007 strain was investigated in order to test the inhibitory effect against the fungi and bacteria of the hospital.

The antimicrobial activity of the endogenous bacterium YC7007 is important in 10 plant pathogenic fungi ( Gibberella fujikuroi , Colletotrichum acutatum (anthrax), Fusarium oxysporum f.sp. cucumerinum (cucumber wilt), Sclerotinia sclerotiorum (scleroderma), Pythium ultimum , Bipolaris oryzae (rice bark pattern bottle), Magnaporthe grisea (rice blast), Botrytis cinerea (gray mold), Botryosphaeria dothidea , Rhizoctonia solani , and four pathogenic bacteria, Burkholderia glumae , Xanthomonas oryzae pv. oryzae (rice blight blight disease ), Pectobacterium carotovorum (bacterial blight disease ), Pseudomonas syringae (flower rot disease) by confrontation bioassay method.

The pathogenic fungi and YC7007 strains were grown in 1 / 5PDA and 1/10 Tcsa on a growth medium (16g agar / distilled water 1 liter) containing 5g of potato agar sugar medium (PDB, Difco) Respectively. A paper disc (diameter 5 mm) was placed at a distance of 1 cm from the edge of the incubator, and 100 μl of the culture broth was thoroughly impregnated on a paper disk. Then, a fungal hyphae mycelium disk (6 mm) And cultured at 24 to 28 ° C for 4 to 6 days.

The bacterial inhibition test was carried out by suspending the bacterial pathogens cultured overnight at 28 ° C. in sterile distilled water (OD ₆₀₀ = 0.1) and 0.1 ml of the suspension was plated on the medium, and the degree of inhibition of growth of the YC7007 strain was measured. The inhibitory effect of the culture filtrate produced by the strain YC7007 on the culture broth was determined by shaking culture (120 rpm) at 28 DEG C for 72 hours in a culture broth except for agar, and the culture broth was centrifuged and filtered with a millipore filter The filtrate was used. In order to examine the response to the temperature of the culture filtrate, the inhibitory effect was examined after 10 minutes of treatment with hot water maintained at each treatment temperature.

Table 2 and Table 3 below show the antibacterial activities of major plant pathogenic fungi of YC7007 strain and major plant pathogenic bacteria.

Referring to Table 2, it can be confirmed that the antimicrobial activity of the endogenous bacterium YC7007 against the phytopathogenic fungus was slightly different depending on the medium, but showed a mycelial growth inhibition effect of 3 to 10 mm.

In addition, referring to Table 3, it can be confirmed that the antimicrobial activity of the endophytic bacterium YC7007 against the plant pathogenic bacteria was 1 / 10TSA, and that the culture solution had a growth inhibition effect of 6 to 10 mm and the culture filtrate had a growth inhibitory effect of 3 to 8 mm.

From these results, it can be concluded that there is a broader pathogen inhibiting effect than any other antagonistic bacteria known to date.

Example  3: YC7007  Test of efficacy of pot experiment for the pathogenic fungi of the strain

Based on the inhibitory effect of the test in the laboratory incubator according to Example 2 above, the effect of controlling the seven major plant diseases of YC7007 strain was investigated as follows. The plant efficacy test was conducted by the Korea Research Institute of Chemical Technology. The target plants were rice blast, rice sheath blight, tomato gray mold, tomato blight ( Phytophthora infestans ), wheat red rust (pathogen: Puccinia recondita ), barley powdery mildew (pathogen: Blumeria graminis f. sp. hordei ) and pepper anthracnose (pathogen: Colletotrichum coccodes .

The treatment was carried out by spraying the suspension of YC7007 strain with the spray gun (1 kg / cm 2) evenly over the entire plant while rotating 2 pots of plants per plant bottle one day before inoculation on a rotating disc. YC7007 strain suspension treatment was conducted by investigating the disease incidence after cultivating in a greenhouse for 1 week at a rate of 5 ml per 3 pots of each host plant 7 days before the inoculation of the strain. The suspension was prepared by shaking culture in 1 / 10TSB medium at 28 ° C overnight, centrifuging the suspension, suspending the bacterial cells in 10 mM MgSO ₄ solution, and adjusting the concentration to 2 × 10 ⁶ cfu / ml. The inoculation and onset of the pathogen were carried out by inoculating the host plants with the spores and the cells of seven pathogens on the plants treated with the strains. Specific experimental methods for each plant disease are as follows.

Rice blast (RCB) M. grisea KJ201 strain was inoculated on a rice bran agar medium and incubated for 2 weeks in a 25 ° C. incubator. The spores formed were harvested with sterilized distilled water to prepare a spore suspension (3 × 10 ⁵ spores / ㎖) Respectively. It was sprayed enough to flow it down to Nakdong Rice (3rd ~ 4th leaf). The inoculated rice plants were incubated for 24 hours in a humid condition and dark for 24 hours. After incubation for 4 days in a temperature and humidity room with a relative humidity of 80% or higher and a temperature of 26 ° C,

Rice sheath blight (RSB) was obtained by inserting an appropriate amount of bran in a 1 liter culture bottle and sterilized, and then the pathogenic bacterium R. solani AG1 The strain was inoculated and cultured in a 25 ° C. incubator for 7 days. For inoculation, the cultured mycelium was finely divided into small pieces, and 4 to 5-leaf Nakdong rice plants treated with the same treatment were uniformly inoculated into the growing pots and cultured for 7 days in a wet condition (25 ° C) sheath) were examined.

Tomato gray mold (TGM) was obtained by inoculating B. sinerea , a pathogenic bacterium, into a potato agar medium and culturing in a constant temperature incubator at 25 ° C. For the inoculation, the spores were harvested and the spore concentration was adjusted to 3 × 10 ⁵ spores / ㎖ using a hemocyte, and then sprayed on the tomato seedlings (2 to 3 leaves) treated with the medicines. The inoculated tomato seedlings were placed in a wet condition (relative humidity of 95% or more) at 20 ℃ for 3 days to induce onset of disease.

Tomato late blight (TLB) is to form a yujujanang cultured at 20 ℃ culture medium was inoculated pathogen pie Saratov shoot inpe Stan's (P. infestans) strain on oatmeal medium. The pathogenic bacteria were harvested by adding sterilized distilled water and the spore suspension with a spore concentration of 3 × 10 ⁴ sporangia / ㎖ was prepared. These were placed in a refrigerator and subjected to low temperature treatment to spill the host to prepare a suspension of the host. This was sprayed on the treated tomato seedlings (2-3 seedlings). Tomato seedlings inoculated with the pathogens were treated at 20 ℃ for 2 days in a wet condition, and the percentage of lesion area on the leaves after 3 days of inoculation was examined in a temperature and humidity chamber.

Since P. recondita , a pathogen of wheat red rust (WLR), is an active parasite, the subspecies formed in wheat seedlings were used as an inoculum while subculturing directly to plants in the laboratory. For the investigation of the efficacy of the strains, five seedlings (diameter: 4.5 ㎝) were seeded with wheat seeds (varieties: silver pine), and the plants were treated with the first seedlings grown in the greenhouse for 8 days. One day after seeding, lt; / RTI > The inoculated wheat seedlings were transferred to a constant temperature and humidity chamber at 20 ℃ with relative humidity of 70% for 1 day in a wet condition at 20 ℃.

Since B. graminis f. Hordei , a pathogen of barley powdery mildew (BPM), is an active parasite, spores formed in barley were used as an inoculum while subcultured in barley seedlings in a laboratory. The medicinal plants were sprayed with barley seeds (diameter: 4.5 ㎝) with 5 lips of barley seeds (variety: Dongbori), sprayed on the first seedlings grown in the greenhouse for 8 days, air dried in the greenhouse, Powdery mildew spores were inoculated. The inoculated barley seedlings were placed in a temperature and humidity room at 20 ~ 23 ℃ and 60% relative humidity for 7 days.

The red pepper anthracnose (RPA) was prepared by inoculating a pathogenic bacterium, C. coccodes , into an oatmeal medium, culturing it at 25 ° C. for 10 days, harvesting the spores formed, adjusting the spore concentration to 2 × 10 ⁵ spores per ml To prepare a spore suspension. Spore suspension was sprayed on the red pepper seedlings (3 ~ 4th leaf stage) treated with the medicines. The spore suspension was inoculated in a wet condition (25 ℃) and treated for 2 days in a constant temperature and humidity chamber (25 ℃, 75% RH). Three days after inoculation, the percentage of lesion area formed on the leaf of pepper was examined.

The control agent was calculated from the lesion area ratio obtained from the bottle irradiation according to the following formula (1).

Table 4 below shows the results of controlling the major plant diseases of YC7007 strain.

As shown in Table 4, the control effect of 7 major plant diseases was 70 ~ 83% in case of rice blast, wheat red rust, barley powdery mildew and pepper anthracnose, but the control effect of rice sheath blight, And in the case of fungal diseases and tomato blight, the control value was lower than 36%. In particular, the control effect of inducing postharvest resistance in soil was only 35% in case of rice, indicating that YC7007 strain has host specificity for rice.

Example  4: YC7007  Rice by inducing disease resistance of strain Egg wilt  Control efficacy test

In order to test the efficacy of YC7007 strain against rice blight caused by the induction of disease resistance, the control effect was investigated as follows.

The rice seedlings (Dongjin 1) seedlings cultivated for 5 weeks in the plant growth phase (28 ~ 30 ℃, relative humidity 80%) were transferred to the sterilized or non sterile rice growing soil and immediately 10 ml of the strain suspension (2x10 ⁷ cfu / (120g). YC7007 strain suspension prepared is 1 / 10TSB medium for one day to 28 ℃ night and then shaking culture was centrifuged bacterial cells 10mM MgSO ₄ Lt; / RTI > solution and adjusting the concentration accordingly. Five days after treatment, a suspension of B. glumae (5 × 10 ⁷ cfu / ml, OD ₆₀₀ = 0.1) was inoculated onto 20 leaves in three replicates on each leaf. The pathogenic bacteria suspension was cultured in R2A medium for 24 hours, centrifuged, and suspended in 10 mM MgSO ₄ solution. The lesion necrosis was investigated after 5 days of inoculation. The disease severity was classified into 0 to 3 (0: no symptoms, 1: small necrotic spots, 2: several necrotic spots were combined to form large nodules, and 3: total necrosis), and the control was calculated by calculating the degree of inhibition . Benzothiadiazole (BTH) (1 mM solution) was used as a control agent for inducing disease resistance in rice.

Table 5 shows the results of controlling the rice blight control effect by the soil treatment of the YC7007 strain, and FIG. 4 is a comparative photograph showing the treatment effects for controlling the blight resistance to rice blight.

As shown in Table 5 and FIG. 4, when the suspension of the strain YC7007 was treated 5 days after the treatment of the soil, the effect of the inoculation of the germs was investigated. As a result, The lesion length was 17.4 mm and the onset was much smaller than 2.4. The control value was 54%, which is equivalent to 57% of the control drug, BTH.

Example  5: YC7007  Effect of inducing disease resistance of the strain to control the blight of rice blast fungus

In order to test the efficacy of YC7007 strain against the blight of rice blight caused by the induction of disease resistance, the control effect was investigated as follows.

The rice seedlings (Dongjin 1) seedlings cultivated for 5 weeks at the plant growth stage (28 ~ 30 ℃, relative humidity 80%) were transferred to the sterilized or non sterile rice cultivation soil and immediately harvested 10 ml of the strain suspension (2x10 ⁷ cfu / Was dispensed into the soil 120g. After 5 days of treatment, the rice blast fungus ( X. oryzae pv. oryzae suspension (1 × 10 ⁷ cfu / ml, OD ₆₀₀ = 0.1) was inoculated on 20 leaves in 3 replicates on each leaf. The suspension of rice blossom blight bacterium was prepared in the same manner as the above rice blight bacterium. After 5 days of inoculation, the necrosis of the lesion was investigated. The degree of disease was classified as 0 to 9 (0: no symptoms, 1: small necrotic spots, 2 to 4: several necrotic spots were merged, middle distangulation, 5 to 8: several necrotic spots were merged to form large necrosis, , And the control was calculated by calculating the degree of inhibition against the control.

Table 6 shows the results of the control of rice blast fungus control effect by soil treatment of YC7007 strain, and FIG. 5 shows comparative photographs showing the effect of controlling rice blight blight disease.

As shown in Table 6 and FIG. 5, after 5 days of treatment with the suspension of YC7007 strain, there was a slight difference according to the pin prick and clipping inoculation method However, YC7007 strain showed 2.3% and 2.2%, respectively, of 69% and 61% (average 65%), respectively, compared with 7.4% and 5.67% of leaves treated with pathogens, respectively. .

Example  6: YC7007  Rice by inducing disease resistance of strain Kidari bottle  Control efficacy test

In order to test the efficacy of the YC7007 strain for controlling rice disease by inducing disease resistance, the control effect was investigated as follows.

The rice seedlings (Dongjin 1) seedlings cultivated for 2 weeks in the plant growth phase (28 ~ 30 ℃, relative humidity 80% or more) were transferred to sterilized or non sterile rice cultivation soil and immediately cultivated in the strain suspension (10 ⁵ to 10 ⁷ cfu / ) Were dispensed to the ground (150 g). Three days after the treatment, G. fujikuroi ( ⁵ × 10 ⁵ cfu / g) was inoculated to each 10 seedlings at 1.5 g / pot. After 10 to 30 days of inoculation, they were placed on the growth plate and the degree of disease was examined. The degree of disease was classified into 0 to 5 (0: no symptoms, 1: slight sulphation of leaves and stem densities, 2 to 3: leaf sulphation and stem kinking, 5: total necrosis) Respectively.

Table 7 below shows the results of investigating the effect of the YC7007 strain on the control of rice hind legs by soil treatment.

As shown in Table 7, when the suspension of YC7007 strain was treated on the soil 3 days after the inoculation of the germs, the degree of disease was 3.4 in the case of the treatment with the pathogen only, but the control value of the strain was 58.8% And excellent resistance to host disease resistance was confirmed.

Example  7: YC7007  Growth Promotion Effectiveness of Rice by Strain Treatment

In order to test the growth promoting activity of rice by YC7007 strain treatment, the plant growth promoting effect was examined as follows.

The rice plant cultivation Sao 1㎏ strain suspension (2x10 ⁷ cfu / ㎖) were inoculated to the rice seed germination after 200㎖ and evenly mixed. Stem and root growth of the plants were investigated after 10 days (seedling stage), 30 days (seeding stage) and 75 days (heading stage), respectively, during the cultivation in plant growth condition (28 ~ 30 ℃ and relative humidity 80% The control soil was mixed with the same amount of 10 mM MgSO ₄ buffer solution alone. Thirty days after the inoculation, 5 ml of the YC7007 strain suspension was treated again to the soil of the rice root to maintain bacterial density at the tillering stage. Each treatment was repeated for 10 weeks for 3 repetitions.

Table 8 below shows the results of the rice plant growth promoting activity of YC7007 strain.

As shown in Table 8, YC7007 strain was cultivated in pots and cultivated in pots, and the growth promoting effect of YC7007 strain was examined at the growth stages of 11.7 cm and 2.7 cm 19.3 cm and 7.7 ㎝, respectively, and the stem length increased by 27.4% and the tilted number was 52% even in the freezing phase. In YC7007 strain, the stem length was increased by 10.8% and the number of tiller was increased by 32%. In addition, YC7007 strain was found to be effective in promoting the growth of rice.

Example  8: Production and effects of antimicrobials inhibiting pathogens

To determine whether inhibition of the pathogen by YC7007 strain was due to production of antimicrobial substances, the production of antimicrobial substances was examined in 1 / 10TSB liquid medium. The degree of inhibition of antimicrobial substances was confirmed by measuring the fungal hyphae or bacterial cell growth inhibition distance formed around the paper disk by the disc diffusion method described above. The YC7007 strain was cultivated in a liquid medium at 28 ° C for 72 hours with shaking (180 rpm), centrifuged at 9000 g for 10 minutes, filtered with a millipore filter (0.2 μm), and assayed for antibacterial activity by incubation time.

FIG. 6 is a graph (a) and a photograph (b) showing the antibacterial effect of the culture filtrate of the strain YC7007 by the culture period, and FIG. 7 is a graph showing the antibacterial effect of the culture filtrate of the YC7007 strain by the temperature treatment.

6, the antimicrobial effect of the culture filtrate of YC7007 strain on incubation period was higher than that of B. glume , X. oryzae and G. fujikuroi , The inhibitory effect was the best in the culture filtrate for 60 hours, and the effect decreased rapidly in the culture filtrate for 72 hours.

In addition, referring to FIG. 7, the stability of the culture filtrate to heat was examined. As a result, the antimicrobial activity was maintained until the treatment at 90 ° C, but the inhibitory effect was slightly decreased when the temperature exceeded 100 ° C. As a result, the antimicrobial substance produced by the strain YC7007 is relatively stable to heat.

Example  9: YC7007  Formulation of the strain

For the activity of YC7007 strain and maintenance of density during storage, it was formulated into powder and liquid form as follows.

The bacterial culture obtained by culturing the strain in a large fermenter (1 ton or more) or the cell obtained by centrifugation is mixed with clay minerals such as kaolin, bentonite and peat at a ratio of 1: 100, And then homogeneously pulverized to prepare a powdery solid phase. At the same time, the bacterial cultures were mixed with the clay minerals at a ratio of 1: 100 to prepare a liquid suspension.

As a result of examining the densities of the formulations as described above, it was confirmed that most of the preparations contained more than 10 ⁸ cfu / g, and the liquid suspension prepared by mixing with the clay minerals contained a density of more than 10 ⁸ cfu / ml .

While the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, Such modifications and changes are to be considered as falling within the scope of the following claims.

Korea Microorganism Conservation Center (overseas) KCCM11275P 20120418

<110> Jgreen Inc.          INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Endophytic bacteria Bacillus Methylotrophicus YC7007 and          development of a multifunctional biopesticide and microbial          fertilizer uning same <130> NP13-04055 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1381 <212> RNA <213> Bacillus methylotrophicus <400> 1 acagatggga gcttgctccc tgatgttagc ggcggacggg tgagtaacac gtgggtaacc 60 tgcctgtaag actgggataa ctccgggaaa ccggggctaa taccggatgg ttgtttgaac 120 cgcatggttc agacataaaa ggtggcttcg gctaccactt acagatggac ccgcggcgca 180 ttagctagtt ggtgaggtaa cggctcacca aggcgacgat gcgtagccga cctgagaggg 240 tgatcggcca cactgggact gagacacggc ccagactcct acgggaggca gcagtaggga 300 atcttccgca atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg aaggttttcg 360 gatcgtaaag ctctgttgtt agggaagaac aagtgccgtt caaatagggc ggcaccttga 420 cggtacctaa ccagaaagcc acggctaact acgtgccagc agccgcggta atacgtaggt 480 ggcaagcgtt gtccggaatt attgggcgta aagggctcgc aggcggtttc ttaagtctga 540 tgtgaaagcc cccggctcaa ccggggaggg tcattggaaa ctggggaact tgagtgcaga 600 agaggagagt ggaattccac gtgtagcggt gaaatgcgta gagatgtgga ggaacaccag 660 tggcgaaggc gactctctgg tctgtaactg acgctgagga gcgaaagcgt ggggagcgaa 720 caggattaga taccctggta gtccacgccg taaacgatga gtgctaagtg ttagggggtt 780 tccgcccctt agtgctgcag ctaacgcatt aagcactccg cctggggagt acggtcgcaa 840 gactgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt 900 cgaagcaacg cgaagaacct taccaggtct tgacatcctc tgacaatcct agagatagga 960 cgtccccttc gggggcagag tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag 1020 atgttgggtt aagtcccgca acgagcgcaa cccttgatct tagttgccag cattcagttg 1080 ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg tggggatgac gtcaaatcat 1140 catgcccctt atgacctggg ctacacacgt gctacaatgg acagaacaaa gggcagcgaa 1200 accgcgaggt taagccaatc ccacaaatct gttctcagtt cggatcgcag tctgcaactc 1260 gactgcgtga agctggaatc gctagtaatc gcggatcagc atgccgcggt gaatacgttc 1320 ccgggccttg tacacaccgc ccgtcacacc acgagagttt gtaacacccg aagtcggtga 1380 g 1381

Claims (6)

Bacillus methylotrophicus YC7007 (accession number: KCCM11275P) strain having a plant disease resistance inducing effect. The method according to claim 1,
The plant disease is Bacillus methylotrophicus YC7007 (accession number: KCCM11275P) strain, which is at least one selected from the group consisting of rice blight, rice blast, and rice hind legs. The method according to claim 1,
This strain is trophy kusu (Bacillus of the Bacillus methyl, characterized in that further has a plant pathogen inhibiting efficacy and plant growth promotion efficacy methylotrophicus ) YC7007 (accession number: KCCM11275P). The method of claim 3,
The plant growth promoting efficacy trophy kusu (Bacillus of the Bacillus methyl, characterized in that the plant growth promoting effects on rice plant methylotrophicus ) YC7007 (accession number: KCCM11275P). A microorganism preparation for fertilizer and a pesticide containing the strain of any one of claims 1 to 4 or a culture thereof as an active ingredient. A microorganism preparation for a fertilizer or a biological pesticide, comprising the strain of any one of claims 1 to 4 or a culture fluid thereof as a physiologically active substance exhibiting the above-mentioned effect as an effective ingredient.

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