KR20080045346A - Bacillus subtilis m27 and biological control of sclerotinia rot by using the same - Google Patents

Abstract

A novel Bacillus subtilis M27 strain is provided to control sclerotium disease effectively, thereby being used as a composition for controlling vegetable sclerotium disease biologically. A Bacillus subtilis M27 strain controlling plant pathogenic bacteria is deposited as a deposition no. KACC 91208P. A microbial agent for controlling the plant pathogenic bacteria comprises a fungus body of the Bacillus subtilis M27 strain or a culture solution thereof as an effective ingredient, wherein the plant pathogenic bacteria is selected from the group consisting of Sclerotinia sclerotiorum, Sclerotium cepivorum, Sclerotinia sp., Sclerotinia minor, Fusarium oxysporum, Botrytis cinerea, Rhizoctonia solani, Didymella bryoniae, Alternaria solani, Corynespora cassiicola, Colletotrichum gloeosporioides, and Phytophthora capsici.

Description

Bacillus subtilis M27 and Biological control of Sclerotinia rot by using the same

Figure 1 shows the phylogenetic location using the 16S rDNA sequence of the novel Bacillus subtilis M27 of the present invention.

Figure 2 shows the 16S rDNA nucleotide sequence of the novel Bacillus subtilis M27 of the present invention.

The present invention relates to the field of biological control of phytopathogens, and more particularly, to a new microorganism having excellent antagonistic activity and a microbial agent and a method for preparing the microbial disease during fresh vegetable cultivation using the same.

Recently, interest in biological control of plant diseases is increasing. Biological control is the use of microorganisms or plant extracts to selectively control the target disease without affecting the environment, other plants and killing, instead of using chemically harmful pesticides that are harmful to the environment in response to increasing consumer demand for safe agricultural products.

Korean Patent Application No. 10-1998-0054040 (Accession No. KFCC 11070, KFCC 11071) and Korean Patent Application No. 10-1999-0034872 (Accession No. KCTC 8831P) have a narrow antifungal spectrum problem, and Korean Patent Application No. 10-2002- 0050582 (Accession No. KCTC 10304BP) is a patent for antimicrobial activity in a medium using apricot seeds, and Korean Patent Application No. 10-2003-0079546 (Accession No. KCTC 10535BP) is limited to tomato late blight, cucumber anthrax, barley and cucumber powdery mildew. have. Korean Patent Application No. 10-2001-0029200 (Accession No. KCTC 0983BP) contains a part of mycobacterial bacteria, but the lactose reaction and Voges-Proskauerqksdmd and the most growing growth of lactose and mannitol in the use of carbon sources It is different from the strain of the present invention in that it was good. In addition, Korean Patent Application No. 10-2001-0029204 (Accession No. KCTC 0985BP) differs from the strain of the present invention in that the lactose reaction in the biochemical properties of this strain and the growth of lactose and mannitol are most favorable in the use of carbon sources. There is.

Bacillus as an Foreign Research subtilis GB03 and Bacillus subtilis MBI600 excludes bacillus and Bacillus subtilis QST713 has the characteristics of induction resistance and is different from the strain of the present invention (The Manual of Biocontrol Agents, 2004).

Therefore, there is an urgent need for the development of a strain that can act efficiently on other phytopathogens other than fungal pathogens, and the development of a method of mass culturing them, without harm to plants.

Bacillus subtilis M27 of the present invention is a very useful microorganism that not only has the effect of inhibiting the pathogenesis against various pathogens, including fungal nucleus, which occurs during the growing season, but also has excellent fixation ability in the soil to inhibit the growth and growth of pathogens. . Therefore, when using the Bacillus subtilis M27 of the present invention, it is judged that it is possible to develop a control method capable of effectively suppressing the disease of the crops without harming the human being.

In order to develop a biological control method of vegetable microbial disease as described above, the present invention is to provide a novel Bacillus subtilis M27 that can effectively control the bacterial microbial disease.

In addition, an object of the present invention is to provide a Bacillus subtilis M27 as a composition for controlling vegetable microbial disease and to provide a microorganism control method using the same.

The present invention is to provide a Bacillus subtilis M27 and the control agent and method for controlling the fungal nucleus using the same.

Root-root bacteria were isolated from soil, and bacillus bacillus subtilis M27 strain was selected through bioassay and bacterium was identified through morphological, physiological and chemical properties and DNA profiles comparison. Bacillus subtilis subtilis ) was identified as M27 .

The present invention provides a culture condition of Bacillus subtilis M27 strain. For industrial use, a method of culturing the strain in large quantities in a short time is essential. Bacillus subtilis M27 strain of the present invention grows well on the TSB medium as a culture medium, it is possible to grow in the range of 14.9 ~ 50 ℃, it grows best at 26 ~ 29 ℃. The growth pH range of the Bacillus subtilis M27 strain of the present invention is 4.5-9.5 and grows best around pH 7.0.

The present invention provides a plant pathogen control agent prepared using Bacillus subtilis M27 strain and a method for controlling plant pathogens using the same.

Sclerotinia forming a bacterium as a treatment target bacterium that can be controlled using the Bacillus subtilis M27 strain of the present invention. sclerotiorum , Sclerotinia minor, Sclerotium cepivorum , Sclerotinia sp ., Botrytis cinerea , Rhizoctonia solani and major vegetable pathogens Colletotrichum gloeosporioides , Corynespora cassiicola , Didymella bryoniae , Alternaria solani , Fusarium oxysporum , Phytophotra capsici and the like. Bacillus subtilis M27 strain of the present invention can be controlled by inhibiting the growth of the bacteria.

Bacillus subtilis M27 strain of the present invention in the greenhouse experiment Sclerotinia sclerotinia sclerotinia is 71.6%, Sclerotium minor had a 61.2% control effect and 71.0% suppression of mycosis in lettuce plant cultivation.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are only intended to illustrate the present invention in detail, and the protection scope of the present invention is not limited to the following examples.

<Example>

Example  1. Isolation, Culture and Identification of Strains

end. Separation and Identification

A) Separation Method: In order to separate the microorganism from the root zone of lettuce in Yangpyeong lettuce culture, the roots were ground, heat treated at 80 ° C for 7 minutes, plated in 10% TSA medium, and then cultured and separated.

B) Identification method: The identification of the isolates was carried out by the size, shape and spore location of the isolates, and the physiological investigations were carried out by temperature response, pH and various biochemical experiments. Molecular biological identification was performed by identifying DNA sequences of 16S rDNA by separating DNA to reveal phylogenetic location. Bacillus subtilis was identified.

Morphology and Biochemical Properties of Bacillus Subtilis M27 Item Characteristics Sporangium bulging - Sphere shape ellipsoidal Sphere position Central Length of cell> 3 μm - Growth at 28 ℃ O Growth at 50 ℃ O Growth at pH 6.0 O Growth with 5% (w / v) NaCl O Anaerobic growth - Degradation of starch O Degradation of casein O Gelatin liquefaction O Utilization of citrate w Nitrate reduction O Oxidase activity O Voges-Proskauer reaction O Acid from L-arabinose O Acid from raffinose O Acid from salicin O Acid from galactose - Acid from lactose O Acid from D xylose O catalase activity O

I. Selection and Culture Method

A) Selection method

-Antagonistic investigation: After incubating for 7 days at 20 ° C by replacing the bacterial total and isolated bacteria in Petri dishes in which PDA medium was dispensed, it was selected first by measuring the distance to inhibit the bacterial bacteria.

-Bioassay: Inoculated with pathogens by cultivating 4.0 leaves of lettuce in 50-hole tray pot against strains selected by antagonistic investigation, and after 24 hours, isolated microorganisms were incubated in TSB medium for 24 hours to irrigate 6ml per plant. The state was examined and finally selected strain (M27) excellent in inhibiting mycobacterial disease.

B) Culture method

In order to establish the mass culture conditions of the selected Bacillus subtilis M27 strain, growth according to the type of medium was carried out using a temperature gradient device (Bio-photo recorder, Advantec Toyo Kaisa, LTD.). Bertani medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 1 l distilled water, TSB medium (Tryptic Soy Broth medium: pancreatic digest of casein 17 g, enzymatic digest of soybean meal 3 g, dextrose 2.5 g, sodium chloride 5 g, dipotassium phosphate 2.5 g, distilled water 1 L) and the growth of the bacteria on four culture medium was found to be the best in the TSB medium, the results are shown in Table 2.

Growth of Bacillus subtilis M27 by Culture Medium badge OD value LB 0.771 TSB 1.777 NB 0.460 KB 0.551

In addition, the growth temperature of the selected Bacillus subtilis M27 strain was investigated using a temperature gradient device (Bio-photo recorder, Advantec Toyo Kaisa, LTD), it is possible to grow in the range of 7 ~ 50 ℃ 26 ~ 29 The best growth was at ℃. As a result of examining the optimum growth pH, growth was possible at the range of 4.5 to 9.5, and the optimum pH was 7.0. The results are shown in Table 3.

Growth of Bacillus subtilis M27 Strain by pH pH 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 OD value 0.517 1.629 1.514 1.523 1.642 1.648 1.560 0.630 0.484 0.286 0.380

Example  2. Phytopathogenic bacteria  Mycelial Growth Inhibitory Effect

Sclerotinia causing mycoticosis among phytopathogens To investigate the mycelial growth-inhibiting effect of four mycobacterium fungi including sclerotioum and eight major phytopathogens, cultivation of 5 mm and total M27 strains of phytopathogens cultured in Petri dishes with PDA media in the room After culturing at 7 degrees for 14 days at 20 degrees by measuring the distance inhibiting the growth of phytopathogens was investigated to obtain the mycelial growth inhibitory effect shown in Table 4.

Inhibitory Effect of Mycelial Growth of Bacillus subtilis M27 against Phytopathogenic Bacteria Mycelial growth inhibition level Pathogens + Phytophotra capsici ++ Didymella bryoniae , Alternaria solani , Corynespora  cassiicola, Colletotrichum gloeosporioides , Fusarium oxysporum , Botrytis cinerea , Rhizoctonia solani +++ Sclerotinia sclerotiorum , Sclerotium cepivorum , Sclerotinia sp . ++++ Sclerotinia minor

Note) +, 5 mm or less; ++, 5.1-10 mm; +++, 10.1-20 mm; ++++, 20.1mm or more

Example  3. in greenhouse To fungal pathogens  Control effect

50 Ball Tray port planting lettuce leaf stage to 4.0 gyunhaekbyeong pathogenic fungi (Sclerotinia Inoculated with barley cultured for 6 days in barley medium sclerotium and S. minor ) 2 inoculation per plant, 24 hours later M27 strain incubated in TSB medium for 24 hours to irrigate 6ml per plant at a concentration of 10 ⁸ cfu / ㎖ As a result of investigating the incidence and inhibiting the incidence of mycosis, the control effect was as shown in Table 5.

Inhibitory Effect of Bacillus Subtilis M27 Strain on Lettuce Fungal Diseases in Greenhouses process Sclerotinia sclerotiorum Sclerotinia minor Occurrence Control price (%) Occurrence Control price (%) M27 treatment 12.0 71.6 19.0 61.2 No treatment 42.3 - 49.0 -

Example  4. Lettuce in the packaging To fungal pathogens  Control effect

Microorganism B. subtilis selected from eco-friendly lettuce culture in Yangpyeong To investigate the inhibitory effect of M27 on mycobacterium microbial disease, 10 days after planting red anchovy lettuce on March 24, 2005, 2 barley inoculated with S. sclerotiorum in barley medium and incubated at 20 ° C for 8 days Lettuce was inoculated into the soil per lettuce abandoned. B. subtilis Treatment of M27 was irrigated 100 ml per week of the culture stock cultured in TSB medium. As a control drug, the benomil watering agent was diluted 1,500 times and subjected to three iterations by irrigation with 100 ml per week. The onset was investigated on April 21 and the results are shown in Table 6.

Benmoylating agent was officially registered fungicide (1500 times benomilifying agent) to control the fungal nucleus of lettuce and was treated together to compare the control effect with chemicals. Of course, it is common to control the microbial disease compared with chemicals, but it was conducted to find out the difference. There are not many microorganisms showing.

Inhibitory Effect of Bacillus Subtilis M27 Strain on Lettuce Mycobacterium on Packaging Treatment contents Byeong stock rate (%) Control price (%) B. subtilis M27 one time irrigation 9.0 71.0 Beno Mild Water (1500 times) 2.7 91.2 No treatment 31.0 -

During the cultivation, the plant surface and soil were treated and settled and the density change was examined 5 times at 7 day intervals. To investigate the isolation and density of the selected antagonists, TSA (trypticase peptone 17 g, phytone peptone 3 g, NaCl 5 g, K ₂ HPO ₄ 2.5 g, glucose 2.5 g, agar 20 g, distilled water 1 liter, pH 7.3) Using a medium, take the soil around lettuce for separation of bacteria of Bacillus , dilute 1 g in 9 ml of sterile water and heat the bacterial suspension at 80 ° C for 10 minutes before dilute plate culture. After repeating three times to examine the density of Bacillus genus bacteria and the results are shown in Table 7. As a result, it was maintained 10 ⁷ CFU (/ g soil) for 14 days from the day of treatment, and after that slightly lower 10 ⁶ CFU (/ g soil) until 28 days. In other words, it was shown to suppress the occurrence of mycosis by maintaining a high density compared to no treatment.

Bacillus species density in soil by days after treatment with Bacillus subtilis M27 strain Treatment contents Bacterial Density by Elapsed Days (CFU × 10 ⁵ / g soil) 0 7 14 21 28 B. subtilis M27 one time irrigation 638.7 131.1 115.0 18.3 84.3 Beno Mild Water (1500 times) 17.3 7.9 1.6 2.7 3.9 No treatment 15.4 4.1 2.8 2.6 9.4

Example  5. Bacillus Subtilis  Mycelial growth section of M27 strain Mycosis  Inhibitory effect

In order to investigate the characteristics of bacterial growth inhibition against S. sclerotiorum (S. minor ) of B. subtilis M27 strain, the culture medium cultured in TSB medium of B. subtilis M27 was centrifuged at 5,000 rpm for 20 minutes. The supernatant was separated and again the supernatant was filtered through a filter to completely remove the cells. In addition, some of the supernatant was concentrated using a concentrator and diluted to 1/10, and some cells and the supernatant were sterilized. B. subtilis M27 cells, sterile cells, supernatants, sterile supernatants, 1/10 times of the B. subtilis M27 cells in 3 places at regular intervals were wound on a 88 mm diameter Petri dish with PDA medium Concentrated supernatant, 1,500 times liquid of benomilizing agent, and no treatment were inoculated with 3 µl of TSB medium and treated three times. The treated Petri dishes were incubated for 10 days at 20 ° C. incubator, and then examined for mycelial growth. Table 8 shows the results of investigating the effect on mycelial growth and mycelial formation of mycobacterial pathogens. As a result, the mycelial growth of the two mycobacterial bacteria was inhibited in the mycelium and the supernatant, the sterile supernatant and the concentrated supernatant diluted 1/10, whereas the mycelial growth was not suppressed at all in the sterile cells. In addition, the suppression of the formation of microbial cells against two mycobacterial fungi occurred in the concentrated supernatant treated with 1/10 of the cells, and in the supernatant treated cells, the microbial nuclei could not be formed only when treated with S. minor .

Inhibition of Mycelial Growth and Mycobacterium Formation of S. sclerotiorum and S. minor by Bacillus subtilis M27 Strain Treatment process S. sclerotiorum S. minor Mycelial growth inhibition (mm) Mycosis Mycelial growth inhibition (mm) Mycosis Cell 15.4 - 27.1 - Sterile Cells 0 + 0 + Supernatant 24.7 + 21.0 - Sterile Supernatant 18.6 + 14.9 + Concentrated Supernatant (1/10) 27.4 - 31.5 - Beno Milding Agent (1500) 34.0 + 11.3 + TSB badge 0 + 0 +

Note) +: Bacillus nucleation,-: Bacillus nucleation

To investigate the germination of S. sclerotiorum (S. minor ) of B. subtilis M27 strains, it was immersed for 30 minutes in a culture stock cultured in TSB medium of B. subtilis M27 , TSB medium and no treatment. It was. Asymptomatic plaque formation examination was carried out three times of 10 cells per triangular flask treated at regular intervals in the sand medium and irradiated with fluorescent lamps at 15 ° C. for 12 hours / day and 76 days later. In addition, the direct mycelial germination of mycorrhizal fungi were treated with three repetitions of 10 per per 88mm diameter petri dishes in water agar medium, and then the mycelial germination rate of mycelia after 8 days in a 20 ℃ thermostat. As a result of the investigation, it showed an excellent germination inhibitory effect compared to no treatment. Table 9 shows the results of investigating the effects on the germ-nuclear germination of the fungal nucleus. In other words, direct germination from mycelium to S. sclerotiorum was 30.0%, which was lower than 100% of no treatment. In addition, direct germination from S. minor bacteria to mycelia was 60.0%, which was lower than 100% of untreated cells, and follicular plaque formation in S. minor bacteria was significantly suppressed to 3.0% compared to 70.0% in untreated cells, reducing the density of infectious agents. I was.

Inhibition of direct germination and follicular plaque formation of mycobacterial bacteria of Bacillus subtilis M27 strain Fungi Treatment concentration Mycelial germination rate (%) Pericardial spot formation rate (%) S. sclerotiorum

10⁸ ㎖^-1 One

10 ⁸ ml ^-1 30.0 0b No treatment 100 100a S. minor One

10 ⁸ ml ^-1 60.0 3.0b No treatment 100 70.0b

Example  6. Bacillus Subtilis  Use of nutrition source of M27

Bacillus The use of carbon and nitrogen sources as subtilis M27 strains is the basic data when mass-liquid culture is used to make a product, and there are differences in the characteristics of nutritional availability from other Bacillus subtilis strains. The growth of the carbon and nitrogen sources was investigated as a method of distinguishing them.

The composition of the Bacillus growth medium used in the experiment is shown in Table 10, and the types of carbon and nitrogen sources are shown in Table 11. Growth effect according to the composition of the medium was measured by absorbance at 620nm after 48 hours incubation of microorganisms.

Composition of Bacillus growth medium ingredient Amount of ingredients sucrose 10.0 g K ₂ HPO ₄ 2.5g KH ₂ PO ₄ 2.5g (NH ₄ ) ₂ HPO ₄ 1.0 g MgSO ₄ 7H2O 0.20 g FeSO ₄ 7H2O 0.01 g MnSO ₄ 7H2O 0.007g water 985 ml

Kind of nutrient source Nutrition Kinds Carbon source Ascorbic acid, Cellulose, Dextrin, Fructose, Galactose, Glucose, Glycerol, Lactose, Maltose, Mannitol, Soluble starch, Sorbitol, Sucrose Nitrogen source (NH ₄ ) ₂ SO ₄ , Glycine, L-Asparagine, NH ₄ NO ₃ , Peptone, NaNO ₃ , NH ₄ Cl, KNO ₃ , L-Proline, L-Glutamic acid, (NH ₄ ) ₂ HPO ₄ , Yeast extract, Casein, Urea, L-Arginine , L-Alanine

In the growth according to the type of carbon source, Fructose was the most effective, and Lactose, Mannitol, and Sucrose were the most effective in the culture of Bacillus growth medium.

In the growth according to the type of nitrogen source, 0.5% of the nitrogen source was added to the Bacillus megaterium growth medium, except for the nitrogen source, and cultured. Casein was most effective. Yeast extract, L-Proline, Peptone, L-Alanine showed the best growth.

The present invention isolated and cultured the novel Bacillus subtilis M27 strain, which was deposited with the Korea Center for Agricultural Microorganisms (KACC) was given accession number KACC 91208P, and provides a method for controlling the culture characteristics and mycobacterial disease. Therefore, the present invention can function as a biopesticide against the fungal nucleus disease of various vegetables occurring during the cultivation can prevent problems caused by crop residues of pesticides, and provide a breakthrough biological control method for the preservation of agricultural ecosystem and comprehensive disease control do.

<110> Rural Development Administration <120> Bacillus subtilis M27 and Biological control of sclerotinia rot          by using the same <130> 1 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1461 <212> DNA <213> Bacillus subtilis M27 <400> 1 tacgacttca ccccaatcat ctgtcccacc ttcggcggct ggctccataa aggttacctc 60 accgacttcg ggtgttacaa actctcgtgg tgtgacgggc ggtgtgtaca aggcccggga 120 acgtattcac cgcggcatgc tgatccgcga ttactagcga ttccagcttc acgcagtcga 180 gttgcagact gcgatccgaa ctgagaacag atttgtggga ttggcttaac ctcgcggttt 240 cgctgccctt tgttctgtcc attgtagcac gtgtgtagcc caggtcataa ggggcatgat 300 gatttgacgt catccccacc ttcctccggt ttgtcaccgg cagtcacctt agagtgccca 360 actgaatgct ggcaactaag atcaagggtt gcgctcgttg cgggacttaa cccaacatct 420 cacgacacga gctgacgaca accatgcacc acctgtcact ctgcccccga aggggacgtc 480 ctatctctag gattgtcaga ggatgtcaag acctggtaag gttcttcgcg ttgcttcgaa 540 ttaaaccaca tgctccaccg cttgtgcggg cccccgtcaa ttcctttgag tttcagtctt 600 gcgaccgtac tccccaggcg gagtgcttaa tgcgttagct gcagcactaa ggggcggaaa 660 ccccytaaca cttagcactc atcgtttacg gcgtggacta cgcagggtat ctaatcctga 720 ttcgctcccc acgctttcgc tcctcagcgt cagttacaga ccagagagtc gccttcgcca 780 ctggtgttcc tccacatctc ntacgcattt ncaccgctac acgtggaatt ccactctcct 840 cttgctgcac tcaagttccc cagtttccaa tgaccctccc cggttgagcc gggggctttc 900 acatcagact taagaaaccg cctgcgagcc ctttacgccc aatagattcc ggacaacgct 960 ngccacctac gtattaccgc ngctgctggc acgtagttag ccgtggcttt ctggttaggt 1020 accgtcaagg tgccgcccta tttgaacggc acttgttctt ccctaacaac agagctttac 1080 gatcctgaaa accttcatca ctcacgcggc gttgctccgt cagactttcg tccattgcgg 1140 aagattccct actgctgcct cccgtaggag tctgggccgt gtctcagtcc cagtgtggcc 1200 gatcaccctc tcaggtcggc tacgcatcgt cgccttggtg agccgttacc tcaccaacta 1260 gctaatgcgc cgcgggtcca tctgtaagtg gtagccgaag ccacctttta tgtctgaacc 1320 atgcggttca aacaaccatc cggtattagc cccggtttcc cggagttatc ccagtcttac 1380 aggcaggtta cccacgtgtt actcacccgt ccgccgctaa catcagggag caagctccca 1440 tctgtccgct cgacttgcat g 1461  

Claims (5)

Bacillus subtilis to control plant pathogens, M. 27 (Bacillus subtilis M27 ) strain (Accession No. KACC 91208P). A microbial agent for controlling plant pathogens comprising the cells of the strain of claim 1 or a culture medium thereof as an active ingredient. 3. The plant pathogen of claim 2, wherein said plant pathogen is Sclerotinia sclerotiorum , Sclerotium cepivorum , Sclerotinia sp ., Sclerotinia minor and Fusarium oxysporum, Botrytis cinerea , Rhizoctonia solani , Didymella bryoniae , Alternaria solani , Corynespora cassiicola , Colletotrichum gloeosporioides, Phytophotra Microbial agent, characterized in that selected from the capsici . A method of controlling plant pathogens by treating plants with the strain of claim 1 or the microbial agent of claim 2. The method of claim 4, wherein the plant pathogen is Sclerotinia sclerotiorum , Sclerotium cepivorum , Sclerotinia sp ., Sclerotinia minor and Fusarium oxysporum, Botrytis cinerea , Rhizoctonia solani , Didymella bryoniae , Alternaria solani , Corynespora cassiicola , Colletotrichum gloeosporioides, Phytophotra A method for controlling plant pathogens, characterized in that selected from capsici .

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