KR101163986B1 - Bacillus velezensis g341 strain and method for controlling plant diseases using the same - Google Patents

Abstract

The present invention relates to a Bacillus velezensis G341 strain and a method for controlling plant diseases using the same, wherein the Bacillus bellensis G341 strain of the present invention is rice leaf blight blight, tomato gray mold, tomato blight, wheat red rust. It is very useful for the control of complex plant diseases because it shows strong antagonism against plant diseases such as tomato powdery mildew, pepper anthracnose, tomato green blight, tomato withered disease.

Description

BACILLUS VELEZENSIS G341 STRAIN AND METHOD FOR CONTROLLING PLANT DISEASES USING THE SAME}

The present invention relates to a Bacillus velezensis G341 strain and a method for controlling plant diseases using the same, and more particularly, the strain having an antagonistic activity against plant diseases by producing an antimicrobial active substance and nematicidal substance. The present invention relates to a microbial agent for controlling plant diseases and a method for controlling plant diseases.

If you do not use pesticides to cultivate crops, there is a problem that yields are reduced from 30% to up to 100%. Therefore, it is inevitable to use pesticides to increase yields. However, the misuse of synthetic pesticides, which make up a large number of pesticides, has caused various problems such as soil, water and agricultural pollution, toxicity, disturbance of ecosystems, and emergence of resistant strains. Recently, the use of synthetic pesticides has been reduced more than 40%, especially in OECD countries, and the demand for organic products is increasing rapidly in developed countries with increasing interest in well-being.

Therefore, in recent years, researches are being actively conducted around the world to develop non-toxic biopesticides that effectively protect crops while minimizing the impact on the environment and other biological systems. Biopesticides are largely divided into plants, microorganisms, natural enemies, natural bioactive substances and genetically modified organisms (GMOs). In particular, research on the development of microbial fungicides has been continuously conducted as a major field of plant pathology for the past 70 years, and over 40 products have been developed in the past 10 years. As of 2004, 76 US microbial pesticides and 113 biochemical pesticides have been registered in the US EPA. In Europe, the definition is slightly different from the US, but published by the British Crop Protection Council (BCPC). According to Biocontrol Agents, 3rd edition ", 112 microbial pesticides are registered and marketed, 58 natural pesticides are registered, and 56 biointeractive compounds such as insect pheromones are registered. In particular, 33 plant-derived natural products are registered. Nineteen microbial pesticides and two natural pesticides are registered in Korea.

Gram-positive bacterium Bacillus (Bacillus) in bacteria is a widely researched and commercialized bacteria Pseudomonas (Pseudomonas) in the next, and produce a variety of secondary metabolites that many existing waterways and biological activity in the soil, stable heat and harsh environments It is characterized by forming endospores that are resistant to. Bacillus velogenis strain having such characteristics was developed by Gustafson in 1994 under the trade name Kodiak as a seed and furrow treatment agent of cotton and peanuts (Backman et al., Improving Plant Productivity with Rhizosphere Bacteria, pp. 3-8). In addition, in 2001, Bacillus Velensis and Bacillus amyloliquefaciens bacteria were mixed to release a microbial fungicide called BioYield. In China, many bacteria of the genus Bacillus are widely used as bacteria for improving yields. Bayer, Germany, has developed a soil infection control agent using Bacillus FZB 24. In addition, Taensa in the United States has released a microbial fungicide using the same bacteria.

Meanwhile, the US AgraQuest Co., Ltd. has developed a microbial fungicide using a strain of Bacillus Belegensis QST713 called Serenade. Unlike the products using the Bacillus bacteria, the microbial fungicide is a fungicide that effectively controls the air infectious diseases that mainly occur on the foliar, and is known to control more than 40 kinds of plant diseases such as gray mold, mozzarella and powdery mildew. These antagonistic microorganisms are known to control a variety of plant diseases by various mechanisms such as competition, parasitics, antibiotic action and induction of resistance, and antibiotics produce more than 30 lipopeptides, which include iturin three groups of substances: iturins, plipastins and surfactins (Ritter, Chemical & Engineering News, 81 , 30-35 (2003)).

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

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

Therefore, the inventors of the present invention, the Gram-positive bacillus bacillus G341 strain isolated from ginseng, rice leaf blight, tomato gray mold, tomato blight, wheat red rust, barley powdery pepper, pepper anthrax, tomato green blight, tomato wilted disease and tomato The present invention has been completed by confirming that there are antagonisms against plant diseases caused by a wide variety of bacteria, fungi and nematodes, such as root-knot nematodes, and thus can be used as microbial agents for controlling plant diseases.

It is an object of the present invention to provide a novel Bacillus velezensis strain having antagonistic action against plant diseases.

Another object of the present invention to provide a microbial preparation for plant disease control using the novel strain.

Still another object of the present invention is to provide a plant disease control method using the microbial agent.

In order to achieve the above object, the present invention provides a Bacillus Velensis G341 strain (KCTC 11497BP) having an antagonistic action on plant diseases.

In order to achieve the above another object, the present invention is a plant disease control containing any one or more selected from the group consisting of Bacillus Velensis G341 strain, spores of the strain, culture medium of the strain and extract of the culture medium as an active ingredient It provides a microbial preparation for.

In order to achieve the above another object, the present invention provides a method for controlling a plant disease comprising treating the microbial agent to a plant or the environment.

Bacillus velogenis G341 strain of the present invention is against plant diseases such as rice leaf blight, tomato ash mold, tomato blight, wheat red rust, barley powdery pepper, pepper anthrax, tomato green blight, tomato wilted disease, and tomato root-knot nematode disease. Since it shows strong antagonism, it can be usefully used for the control of complex plant diseases.

Figure 1 is a scanning micrograph of the Bacillus Velensis G341 strain.
Figure 2 shows the phylogenetic tree (phylogenetic tree) by the gyrase A gene sequence of the G341 strain.
Figure 3 is the result of examining the production capacity of the protease, chitinase and cellulase of the G341 strain.
4 shows growth inhibition of various phytopathogens of the G341 strain.
5 is ESI-MS analysis data of the cation mode of the antimicrobial substance 1 isolated from the G341 strain.
FIG. 6 shows MS / MS data of m / z 1035 ion peak, which is one of [M + 1] ⁺ ions from the cation mode ESI-MS analysis of the antimicrobial substance 1 isolated from G341 strain.
7 is ESI-MS data of the cation mode of antimicrobial substance 2 isolated from G341 strain.
FIG. 8 shows MS / MS data of m / z 741.3 ion peak, which is one of the [M + 1] ²⁺ ions from the cationic mode ESI-MS analysis of the hydrolyzate of antimicrobial active substance 2 isolated from the G341 strain.
9 shows the effect of seed coating treatment on the foot blight of tomato using cells of the G341 strain. The plant on the left shows the growth of tomatoes that have been treated with seed coating of the G341 strain, and many plants are still growing normally. The photo on the right shows the growth of untreated tomatoes and almost all plants are dead.
Figure 10 shows the effect on pepper blight of G341 solid cultures. In the untreated group, the disease became more severe over time, and almost all plants withered and died after 19 days, whereas the G341 solid culture treatment group shows little development.

Hereinafter, the present invention will be described in detail.

The present invention provides a Bacillus Velensis G341 strain (KCTC 11497BP) having antagonism against plant diseases. The strain was isolated from the roots of ginseng and the analysis showed the following morphological, biochemical and genetic characteristics:

1) Morphological characteristics: It grows thinly spread on Tryptic soy agar medium, single colony with small snowflake shape, rod (Fig. 1), positive in gram staining, endogenous spore formation It has a peritrichous flagella.

2) Biochemical Properties: Using arabinose, mannitol and xylose to produce acid and also break down starch.

3) Genetic characteristics: The nucleotide sequence of the gyr A gene (SEQ ID NO: 3) shows a homology of about 96.6% to Bacillus velogenis LMG22478 ^T.

From the above characteristics, the strain of the present invention was identified as a new strain belonging to Bacillus velezensis , which is named as Bacillus veregens G341 and dated April 6, 2009, Korea Research Institute of Bioscience and Biotechnology Was deposited as Accession No. KCTC 11497BP.

The Bacillus bellensis G341 strain according to the present invention is characterized by exhibiting strong antagonism against plant diseases. The strain is Magnaporthe grisea , a causative agent of febrile disease , Rhizoctonia solani , a causative agent of rice leaf blight, and phytophthora capcici, a causative agent of pepper blight. ( Phytophthora capsici , Phytophthora infestans , a causative agent of tomato late blight , Botrytis cinerea , a causative agent of tomato gray mold, and Puccinia recondita , a causative agent of wheat rust ), Erysiphe graminis f. Sp. Hordei , a causative agent of barley powdery mildew, Sclerotinia sclerotiorum , a ginseng spot pattern Alternaria panax , a causative agent of deciduous bacteria, and Colletotrichum cocodes , a causative agent of pepper anthrax, were inhibited (Example 5) In addition, the G341 strain of the present invention was root-knot nematode (Meloidogyne incognita), when the pathogen Fusarium oxy-form speaker system sports room Rico Persie wilt (Fusarium oxysporum f. sp. lycopersici ), Chinese cabbage clubroot Plastic causative organism of the SUMO to radio Fora bra said car (Plasmodiophora brassicae), and the LAL Stony Oh Solana Seah room (Ralstonia solanacearum) of the foot blight causative organism was inhibited (Examples 10 to 16).

Therefore, G341 strain of the present invention is rice leaf blight blight, rice blast, tomato gray mold, tomato blight, tomato green blight disease, tomato wilting disease, tomato rootworm nematode disease, wheat red rust disease, barley powdery mildew, pepper anthrax, pepper blight, lettuce It is characterized by exhibiting a strong antagonistic action against any one or more plant diseases selected from the group consisting of fungal nucleus disease, ginseng dot pattern deciduous disease, Chinese cabbage root gall disease, radish rot and cucumber root gall nematode disease.

On the other hand, the present invention provides a microbial agent for controlling plant diseases containing any one or more selected from the group consisting of Bacillus Velensis G341 strain, spores of the strain, culture medium of the strain and extract of the culture medium. . The culture medium of the strain may include all of the culture solution containing the strain or culture medium filtered strain. In addition, the preparation may include a carrier in addition to the G341 strain, the spores of the strain, the culture of the strain and the extract of the culture, water, white carbon, kaolin and the like may be used as a preferred carrier. The culture extract is preferably extracted from the culture solution with ethyl acetate, butanol, water or a mixed solvent thereof. More preferably, the butanol layer or the culture supernatant obtained by dividing the culture supernatant with the same amount of ethyl acetate and butanol with the same amount of ethyl acetate. The remaining aqueous layer may be fractionated with butanol. The preparation may be used by mixing the Bacillus bellensis G341 strain, the spores of the strain, the culture solution of the strain and the extract of the culture solution with a carrier, and then formulated into a powder, pellet, granule or solution.

Furthermore, the present invention provides a method for controlling plant diseases, comprising treating the microbial agent according to the present invention to a plant or the surrounding environment.

The plant diseases include rice leaf blight blight, rice blight, tomato gray mold, tomato blight, tomato green blight, tomato wilting disease, tomato root worm nematode disease, wheat red rust disease, barley powdery mildew, pepper anthrax, pepper blight, lettuce fungus nucleus disease, ginseng dot pattern It may be any one or more selected from the group consisting of deciduous disease, Chinese cabbage root gall disease, radish rot and cucumber root gall nematode disease. The microbial agent may be treated by i) treating the soil, ii) treating the surface of the plant, iii) coating the seed, or iv) in a combination thereof to prevent inhibition or death of plant growth by plant diseases. have. The Bacillus vegetosis G341 strain of the present invention in the formulation is, for example, 1.0 x 10 ⁴ cells / ml to 1.0 x 10 ¹⁰ cells / ml, preferably 1.0 x 10 ⁶ cells / ml to 1.0 x 10 ⁹ cells A solution prepared as described above at a concentration of / ml may be treated in an amount enough to flow down the target plant, but is not limited thereto.

[Example]

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1 Isolation of Strains from Ginseng Root

The ginseng with rot symptoms was divided into small pieces from 4 years old ginseng roots, and then immersed in 1% sodium hypochlorite (NaOCl) for 1 minute and then washed in sterile water for 1 minute. The washed ginseng root tissue was ground by adding 0.1 M magnesium sulfate (MgSO ₄ ) solution, and then plated on potato dextrose agar medium (PDA; Difco, USA). After incubation at 28 ° C., when bacterial colonies appeared, single colonies were isolated by passage 3-4 times in nutrient agar medium (Nutrient agar).

Example 2: Identification of Isolated Strains

For the strain isolated in Example 1, morphological and biochemical properties were analyzed, and genetic properties were analyzed through the gyr A gene sequence.

(1) Morphological and biochemical properties

The strain isolated in Example 1 was grown in a thin spread on tryptic soy agar medium, a single colony showed a small snowflake shape. As a result of observing with a scanning microscope (model name JEM1010; JEOL, Japan), as shown in Fig. 1, the strain was a rod and showed positive in Gram staining reaction. Furthermore, it formed endospores and had peritrichous flagella.

On the other hand, using the Biolog program (Biolog program; Biolog Inc., USA) as a standard method to analyze the substrate capacity of the Bacillus Velensis G341 strain and LMG22478 ^T of the present invention the results are shown in the following table 1 is shown.

As shown in Table 1, the strains of the present invention are positive in availability of D-xylose, para-hydroxyphenylacetic acid, alpha-ketovaleric acid, lactamide, putrescine, adenosine and inosine, whereas the LMG22478 ^T strain is As negative, both strains were found to have completely different biochemical properties.

(2) Genetic characteristics

Colony polymerase chain reaction (colony PCR) was performed to obtain the nucleotide sequence of the gyr A gene of the strain. Collect colonies using a toothpick from a single colony and mix them with PCR-premix (PCR-premix; iNtRON Biotechnology, South Korea) and then p-gyrA-f (5'-CAG TCA GGA AAT GCG TAC GTC CTT- Gyr A gene was amplified using primers 3 '; SEQ ID NO: 1) and p-gyrA-r (5'-CAA GGT AAT GCT CCA GGC ATT GCT-3'; SEQ ID NO: 2). The amplified PCR product was purified using a Wizard PCR prep kit (Promega, Medison, WI, USA), and then commissioned to Macrogen (Daejeon, South Korea) to obtain a base sequence. The base sequence was compared with GenBank database and base sequence using BLAST of NCBI. Sequences were sorted by CLUSTAL X and a neighbor-joining tree was created using PHYDIT program version 3.0.

As a result of analyzing the nucleotide sequence (SEQ ID NO: 3) of the gyr A gene of the strain based on the neighbor-joining algorithm, it was found that the strain is a novel strain having about 96.6% homology with Bacillus velogenis LMG22478 ^T. (FIG. 2).

From the above results, the strains obtained in the present invention were identified as Bacillus Velensis strains, and the strain was named Bacillus Velensis G341, and was assigned to KCTC 11497BP dated April 6, 2009 to the Korea Institute of Bioscience and Biotechnology. Deposited as.

Example 3 Investigation of Enzyme Production Capacity of Bacillus Velensis G341 Strain

It was examined whether the G341 strain produced protease, cellulase and chitinase.

Reputation of 1/10 Luria-Bertani agar (purchased by Difco, USA) containing 1% (w / v) skim milk (purchased by Difco, USA) to determine the production of protease G341 strains were inoculated in a stroke using a loop in the center of the medium, and after 24 hours of incubation at 30 ° C, it was examined whether a clear zone was formed around the colonies.

Chitin agar medium (Colloidal chitin 7g, KH ₂ PO ₄ 3g, K ₂ HPO ₄ 2g, NH ₄ Cl 1 g, MgSO ₄ -7H ₂ O 0.2g, CaCl Inoculated with G341 strain in the center of ₂ ~ 2H ₂ O 0.01g, yeast extract 0.1g, agar 15g and distilled water 1L) and incubated for 3 days at 30 ℃ and examined the formation of a clear zone around the colonies.

Cellulose agar medium (MgSO ₄ ~ 7H ₂ O 0.1g, CaCl ₂ ~ 2H ₂ O 0.2g, KH ₂ PO ₄ 0.3g, K ₂ HPO ₄ 0.5g, Carboxymethylcellulose 5g, Inoculated with G341 strain in the center of 0.1 g of yeast extract, 15 g of Bacto agar and 1 L of distilled water, and incubated at 30 ° C. for 3 days, followed by pouring 1 mg / ml of congo red onto the medium. After leaving for 10 minutes, discarded, rinsed three times with 1 M sodium chloride, and then investigated the size of the orange zone (orange zone) formed around the colony, the results are shown in FIG.

As shown in FIG. 3, the G341 strain produced proteases and cellulase, but did not produce chitinase.

Example 4: Determination of phytopathogenic growth inhibitory activity of Bacillus Velensis G341 strain

In order to examine the effect of the Bacillus Velensis G341 strain isolated in Example 1 on the growth of phytopathogens, growth inhibition activity was measured in 9 representative phytopathogens (Table 2). Specifically, the mycelia of the phytopathogens shown in Table 2 obtained from the cultivated cultures were inoculated in the center of PDA medium. Thereafter, the G341 strain was plated in a straight line at a distance of about 2.5 cm from the inoculation site, and then cultured at 25 ° C. When phytopathogens grow to the edge, the growth inhibition of phytopathogens by G341 strain was measured and the results are shown in Table 2 and FIG. 4.

Growth Inhibitory Activity of Phytopathogenic Bacteria of G341 Strains Plant pathogen Scientific name Low land length (mm) Cucumber mozalok disease Pythium ultimum 2 Rice leaf blight Rhizoctonia solani 10 Tomato wilt Husa Solarium Room Spokane oxy form RY beaded sheath Rico City (Fusarium oxysporum f.sp. lycopersici) 10 Pepper Plague Phytophthora Capsici
( Phytophthora capsici ) 17 Lettuce fungi Screrotine Scleroroom
( Sclerotinia sclerotiorum ) 21 Pepper anthrax Collectortreecum Cocodes
( Collectotrichum coccodes ) 16 Rice Bacteria Magnaporte Grisséa
( Magnaporthe grisea ) 20 Ginseng spot pattern deciduous bacterium Alternaria panax 16 Tomato gray mold Botrytis cinerea 13

As can be seen in Table 2 and Figure 4, G341 strain according to the present invention was shown to strongly inhibit the growth of all phytopathogens except cucumber mozzarella ( Pythium ultimum ). One peculiarity is that it has excellent growth inhibitory activity against Phytophhotra capsici , which belongs to the fungus coliform fungi. To date, no bacterium exhibiting antimicrobial activity against red pepper bacterium among bacillus strains is believed to be the first strain of the present invention.

Example 5 Preventive Effect on Seven Plant Diseases by Leaf Spray Spray

Inoculation of causative organisms of rice leaf blight, tomato gray mold, tomato blight, wheat red rust, barley powdery mildew and caustic anthracnose causative organism to examine the preventive effect against plant diseases of Bacillus velensis G341 strain isolated in Example 1 The prophylactic effect of G341 treatment was investigated the other day. Experimental method was sprayed on the cultivated culture of Bacillus Velensis G341 strain to rice, tomato, wheat, barley and red pepper, and then inoculated with each pathogen after 1 day to control Bacillus Velensis G341 strain. The activity was investigated.

Specifically, the Bacillus velogenis G341 strain was inoculated in a sterilized 200 ml Tryptic soy broth (Tryptic soy broth, purchased from Becton and Dickinson) and then incubated at 150 rpm for 3 days at 150 rpm. 100 ㎍ / ml xanthan gum was added to 30 ml of the diluted solution diluted 10-fold with distilled water, and then treated with rice, tomatoes, wheat, and barley, respectively, and dried for 1 day at room temperature. . On the other hand, rice, tomatoes, wheat and barley used in the experiment, before filling the G341 strain, filled with water or gardening soil 70% in plastic pots with a diameter of 4.5 cm, sowing seeds and greenhouses at 25 ± 5 ℃ Cultivation for 1 to 4 weeks. Rice leaf beetle blight was inoculated in 5-leaf seedlings by incubating the culture obtained from Rhizoctonia solani (Korea Research Institute of Chemical Technology) for 7 days in medium (90 g of wheat bran, 15 g of chaff and 100 ml of distilled water). After wet treatment for 4 days in a wet room, the incubation was induced by incubation for 4 days in a constant temperature room at 25 ℃. Tomato plague was sprayed with 3 ~ 4 leaf tomato seedlings and sprayed with the saponified suspension extracted from the jujube sac (10 ⁵ jujube sac / ml) of Phytophthora infestans (Gangneung University). In fact, 2 days of wet treatment and incubation for 1 day in a constant temperature and humidity room of 25 ℃ induced the onset. Tomato gray mold disease was treated with spore suspension (10 ⁶ spores / ml) of Botrytis cinerea (Korea Research Institute of Chemical Technology), a causative agent of gray mold disease, in tomato seedlings of 3-4 leaves. In fact, 3 days incubation was induced to induce. Wheat red rust sprays spores of Puccinia recondita (University of Incheon), the causative agent of rust, known as biolactic bacteria, in a single-leaf seedling, suspended in 250 µg / ml Tween 20 solution with 0.67 g spores / l. After treatment for one day in a 20 ℃ humid room and transferred to a 20 ℃ constant temperature room was incubated for 6 days to induce the onset. Barley powdery mildew was inoculated by spraying spores of Erysiphe graminis f. Sp. Hordei (Korea Research Institute of Chemical Technology), a causative agent of powdery mildew, passaged from host plants, on seedlings of barley . Incubation was induced by incubation for 7 days in a constant temperature room. The lesion area ratios were investigated 7 days after inoculation for wheat rust and wheat flour, 8 days after inoculation for rice leaf blight, and 3 days after inoculation for tomato gray mold and tomato late blight.

On the other hand, for the control activity test for pepper anthracnose disease, 70% of gardening topsoil is filled in a plastic pot with a diameter of 7.0 ㎝, sowing the seeds of the most peppers, grow them up to 3-4 leaves in the greenhouse and then prepared Bacillus Cultures of the Velensis G341 strain were foliarly sprayed. After spraying the sample was dried at room temperature for 24 hours, and then spray inoculated with a spore suspension (4 x 10 ⁵ spores / ml) of the pepper anthrax pathogen , Colletotrichum coccodes (Korea University). After 2 days of invasion in a humid room, it was left in a constant temperature room at 25 ° C. for 1 day, and the lesion area ratio was examined after 3 days of inoculation. The lesion area ratio was examined by Moon, and the control value was calculated according to Equation 1 using the obtained lesion area ratio and the results are shown in Table 3 below.

[Equation 1]

As shown in Table 3, the Bacillus Velensis G341 strain of the present invention was shown to exhibit excellent control against rice leaf blight blight, tomato gray mold, tomato blight, wheat rust, barley flour and pepper anthrax.

Example 6: Nematode Activity against Root-knot Nematodes of Bacillus Velensis G341 Strains

The nematicidal activity against the tomato root-knot nematode ( Meloidogyne incognita ) of the Bacillus velogenis G341 strain was investigated. G341 strains were inoculated into BHI (Brain Heart Infusion) liquid medium (Difco, USA) and then shaken for 30 days at 30 ℃, 150 rpm. The culture was centrifuged at 8000 xg for 10 minutes and then the supernatant was taken. To investigate the heat stability of the nematicidal active ingredient, 50%, 10%, 5%, 2%, 1%, 0.5%, 0.2%, and 0.1% heat-treated samples at 100 ° C. for 20 minutes and unheated samples were used. After dilution with% and 0.01%, root-knot nematodes were treated by the above concentrations. Root-knot nematodes used in the experiments were isolated from infected areas of tomatoes. That is, the soil was completely removed from the roots using water, and then the egg masses of the root-knot nematodes were separated using tweezers. The separated ovum was washed with sterile water and then stirred in 0.5% sodium hypochlorite (NaOCl) solution, filtered through a 26 μm sieve, and washed with sterile water. The eggs were hatched for 3 to 5 days and then second-larva larvae were obtained using the Baerman funnel method. 500 μl of culture supernatant was added to each well of a 96-well plate and 100 larvae were placed therein. The untreated section was treated with sterile water containing BHI medium components. After 24 hours at 25 ° C., the number of live and dead nematodes was measured. The experiment was performed in 5 repetitions, and the same experiment was repeated twice, and then averaged. The measurement results are shown in Table 4 below.

As shown in Table 4, the culture supernatant of G341 showed very potent nematicidal activity against larvae of root-knot nematodes. To date, there have been no reports showing that Bacillus bellensis strains show nematicidal activity, which is a very specific result.

In addition, the heat-treated supernatant also showed almost the same nematicidal activity, which means that the component showing nematicidal activity is very stable against heat.

Example 7: Isolation and Identification of Antimicrobial Active Ingredients

In order to isolate the antimicrobial material produced by the Bacillus Velensis G341 strain, the strain was inoculated into 200 ml of Tryptic soy broth (purchased by Becton and Dickinson, USA) and then inoculated at 30 ° C. for 3 days. Shake culture at 150 rpm. The culture solution was centrifuged at 8000 xg for 10 minutes to obtain a culture supernatant (8 L), which was fractionated twice in succession each with the same amount of ethyl acetate and butanol. The obtained butanol layer was concentrated under reduced pressure to obtain an extract of 17.25 g. One half of this was added to a silica gel column and eluted with chloroform: methanol: water solvent (30: 9: 1 and 65: 25: 4). Among them, fractions showing inhibitory activity against phytopathogens as in Example 4 were collected and concentrated under reduced pressure to obtain an eluate of 5.46 g. Each fraction obtained through the silica gel column was dissolved in methanol at a level of 10 mg / ml, and then, 35 µl each was dipped onto a paper disc (diameter = 8 mm), dried and killed by inoculating rice medium with PDA. Cultures at 25 ° C. were used to specify the diameter of the lowland area exhibited by mycelial growth inhibition. For the untreated group, 35 μl of methanol was treated. 3 g of this was added to a Sephadex LH-20 column, eluted with 100% methanol, only the active fractions were concentrated under reduced pressure, and then added to a C18 reversed phase column. The column started eluting with 100% water and finally eluting with 100% methanol with increasing methanol content. In this way, two active substances (substance 1 and substance 2) were separated.

MS / MS analysis was performed to determine the structure of the separated material. The instrument used a Hybrid Quadrupole-TOF LC / MS / MS mass spectrometer (AB Sciex Instruments, Calif., USA) and analyzed in electrospray ionization (ESI) positive ion mode. For Material 1, as a result of ESI-MS analysis, the [M + 1] ⁺ ion peak was shown at 1035 as shown in FIG. 5. As a result, MS / MS data were obtained for the 1035 peak, which is the main peak. As shown in FIG. 6, it was shown that it was composed of Asp-Tyr-Asn-Ser-Gln-Ser-Thr (SEQ ID NO: 4). (Bacillomycin) L was identified.

ESI-MS analysis was carried out in cation mode to identify the structure of material 2, and as shown in FIG. 7, the [M + 1] ⁺ ion peak was found at 1478. The ion peaks appearing between m / z 725 and m / z 763 are the peaks represented by [M + 1] ²⁺ ions. To obtain MS / MS mass spectrum of this material, material 2 was hydrolyzed with KOH, and again MS / MS data was obtained for 741.3 ion peak, one of the appearing [M + 1] ²⁺ ions. As a result, as shown in Figure 8 Glu-Orn-Tyr-Thr-Glu-Ala-Pro-Gln-Tyr-Ile (SEQ ID NO: 5), it was identified as pengycin (fengycin) A.

The two antimicrobial substances isolated were examined for the prevention effect 1 day before the inoculation in the same manner as in Example 5 in six plant diseases. Rice blast is Won Kyun rice blast (Magnaporthe seedlings in the third leaf stage gisea , obtained from: Seoul National University) spore suspension (5 x 10 ⁵ spores / ㎖) was inoculated by spraying and then incubated in a 25 ℃ incubated for one day, and then incubated in a 25 ℃ constant temperature room for 5 days. The measurement results are shown in Table 5 below.

As shown in Table 5, Basillomycin L showed control against rice blast, rice leaf blight and pepper anthrax, while Pengisin A showed rice leaf blight, tomato ash mold, wheat red rust and barley powdery mildew. It showed a control effect against. Therefore, the G341 strain of the present invention may be effectively used for controlling the plant diseases.

Example 8: Identification of Nematicide Active Ingredients

In order to isolate nematicidal active ingredient produced by the Bacillus velogenis G341 strain, the culture supernatant was extracted twice in succession with the same amount of ethyl acetate and butanol. The two organic solvent layers and the aqueous layer were concentrated under reduced pressure, respectively, and then the in vitro activity of the nematode nematode was measured against tomato root-knot nematodes by concentration. The measurement results are shown in Table 6 below.

As can be seen in Table 6, the aqueous solution layer was found to be the most active. This indicates that the active ingredient is not extracted with ethyl acetate and butanol and is very water soluble. On the other hand, when added to the Sep-Pak C18 cartridge (Supelco, USA), the active ingredient did not pass through the column, the active ingredient was estimated to be a protein of high molecular weight.

Quihong et al. (Quihong, Applied Microbiology and Biotechnology , 69 (6): 722-730, 2006) describe an extracellular cuticle degradation protease with significant similarity to the subtilisin BPN ′ produced by the B16 strain of Bacillus. It has been reported that degrading protease kills Panagrellus revivivus nematodes. Thus, the G341 strain was also treated with protease inhibitor cocktails (protease inhibitor cocktails: P8465, Sigma-Aldrich, USA) in order to confirm that the G341 strain also produced protease and showed nematicidal activity.

Specifically, 1 ml of DMSO (dimethylsulfoxide) was added to P8465 (5 ml), followed by stirring for 1 minute, and then 4 ml of distilled water was added to prepare a protease inhibitor solution. Wherein P8465 is 23 mM AEBSF (4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride), 0.3 mM pepstatin, 2 mM bestatin A, 100 mM EDTA and 0.3 mM E- 64 protease inhibitors. In addition, BHI medium and Tris-HCl (pH 8.0) buffer were added at the same concentration as the sample as an untreated control.

The protease inhibitor solution prepared above was added to the culture supernatant in 10% content, and the samples were diluted to 1%, 0.5%, 0.2%, 0.1% and 0.05%, respectively. It is shown in Table 7 below.

As can be seen in Table 7, the nematicidal activity of the culture supernatant of the G341 strain was almost suppressed by the protease inhibitor. Thus, the main nematicidal active ingredient of the G341 strain was found to be protease.

Example 9: Gene Isolation and Sequence Analysis of Nematicidal Active Protease

The following forward and reverse primers were prepared by comparing the conserved regions of homologous proteases with the Bacillus genus B16 alkaline serine protease (AY708655) reported to have nematicidal activity:

SPB16_F: GTGAGAGGCAAAAAGGTA (SEQ ID NO .: 6)

SPB16_R: CTGAGCTGCCGCCTGTAC (SEQ ID NO .: 7).

After purifying chromosomal DNA of G158 strain, PCR was performed with the primers. After 5 minutes of reaction at 94 ° C., the reaction of 30 seconds at 98 ° C., 30 seconds at 55 ° C. and 1 minute at 72 ° C. was repeated 30 times. Finally, the reaction was carried out at 72 ° C. for 2 minutes. The PCR product was purified, linked to the pGEM-T Easy (promega) vector, and transformed into Escherichia coli DH5α. After plasmid (G158SP GEMT EASY) was purified from the E. coli, the sequencing was performed using the following T7 and SP6 primers.

T7 primer: AATACGACTCACTATAG (SEQ ID NO .: 8)

SP6 Primer: ATTTAGGTGACACTATAG (SEQ ID NO .: 9)

The nucleotide sequence of the gene encoding the nematicidal active protease of the G341 strain was found to have the nucleotide sequences of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, and the B16 alkaline serine protease and the base of Bacillus genus. 98% homology at the level confirmed that the subtilisin (subtilisin) gene.

Example 10: Root-knot nematode control effect by G341 strain

In the culture medium of G341 strain, the control effect against tomato and cucumber root nematode disease in greenhouse was investigated. Plant seedlings grown for 1 week to 10 days after sowing tomatoes and cucumbers were transplanted to the soil infected with root-knot nematodes. The diameter of the pot was 6.6 cm and the amount of topsoil per pot was about 140 ml. The solution diluted with the stock solution and distilled water of the culture solution of strain G341 was irrigated 30 ml per pot. Meanwhile, as a positive control group, a strong pesticide nematode (150 mg / ml, obtained from Dongbu HiTek) was treated. After the treatment, it was grown for one month in a greenhouse and then examined for incidence. Depending on the number of nodules formed on the roots, the incidence is indicated as 0, 1, 2 and 3. The experiment was repeated five times per treatment, and the control effect against tomato and cucumber root-knot nematode diseases is shown in Table 8 below.

As can be seen in Table 8, the G341 broth stock solution showed 80% and 67% control activity against tomato root knot nematode disease and cucumber root knot nematode disease, respectively, and 50% of 1/10 dilutions of the culture medium, respectively. And 40% control activity. From the above results, although the effect was lower than that of the synthetic nematicide nematode nematode, it was found that the G341 culture of the present invention is effective in the control of root-knot nematode disease according to the control effect of up to 67%.

Example 11 Analysis of Effect of Seed Coating on Germination Rate

The G341 strain was shaken in BHI liquid medium at 150 rpm and 30 ° C. for 2 days, and then white carbon (20 g) and dextrin 100 (50 g) were added to 1 L of the culture, followed by spray drying. The spray drying was using a spray dryer of EYELA company, the inlet temperature is 165 ℃, the outlet temperature is 64 ℃, the feeding rate is 650 ml / hr (3.0), the blower (blower) 0.3 It was carried out at -0.4 m ³ / min. After spray drying, the cfu of the spray dried product was measured. That is, the sample was diluted with sterile water and then plated on nutrient agar medium (nutrient broth), and then cultured at 30 ° C. to determine the number of colonies formed. The measurement produced 4 x 10 ¹⁰ cfu / g colonies. On the other hand, 37.5 g of the spray-dried sample was mixed with 1.24 kg of a subsidiary used for seed coating, and seed coating was performed on seed and 20,000 grains of Seogwang tomato seed. The seed was coated in horticultural soil and soil, and the germination rate was measured. The measurement results are shown in Table 9 below.

As can be seen in Table 9, the germination rates of the seeds coated with the G341 strain and the uncoated seeds were almost the same. As a result, it was found that the G341 strain had little effect on the germination of seeds.

Example 12 Control Effect of Tomato Root-knot Nematode and Withered Bacteria on Complex Infected Plant Disease by Seed Coating in Greenhouse

Example 11 After the seed coat the tomato, such as, complex infection of Fusarium oxy sports rooms form RY cis Rico beaded during (Fusarium oxysporum f. Sp. Lycopersici ) , and root-knot nematode (Meloidogyne incognita) the causative agent of root-knot causative agent of wilt The control effect on was investigated.

Specifically, 500 g of sterile sand and a topsoil mixture were filled in a 6 cm diameter pot, and the tomato seedlings grown for 3 weeks were transplanted. Each plant was inoculated with the pathogenic fungus and root-knot nematodes simultaneously. In the case of fusiform bacteria, 20 g of medium incubated in chaff medium for 15 days was mixed with 5 cm of the soil top of the pot. Root-knot nematodes were inoculated with 1,000 larvae per second. 5 treatments were repeated and the same experiment was repeated. After 7 weeks of inoculation, the plants were pulled and the soil was removed from the roots, and the incidence was examined.

Incidence is defined as 0: disease free, 1: 1 leaf sagging or yellowing or withering, 2: 2 and 3 leaves yellowing or withering, 4: whole plant yellowing or withering, 5: whole plant completely dead And the control value thereof was measured and the results are shown in Table 10 below.

As shown in Table 10, the coated seeds were 67% and 56%, respectively, for the control of wilt and rot nematode disease. On the other hand, when irrigation of the culture stock solution did not appear at all withered symptom, only root-knot symptom appeared. Therefore, it is expected that the plant disease may be effectively controlled when the seed coating and the culture irrigation are used together in the case of tomato Fusidium wilting disease and root-knot nematode disease.

Example 13: Control effect of tomato foot blight by seed coating

Seeds coated with the G341 strain and uncoated seeds were sown in a greenhouse, grown for one month, and then planted in a plastic house in Cheongju, Chungcheongbuk-do. After 25 weeks, two meals were prepared. After 60 days, the number of living and dead tomato plants was counted. In case of the soil, tomato foot blight was severely developed every year, but soil analysis showed that there was almost no root-knot nematode. Instead, the diseased plant stems were collected from the disease and identified as bacteria. As a result, it was identified as Ralstonia solanacearum , which is a foot blight bacterium. . Effect of seed control using seed coating using G341 strain is shown in Table 11 and FIG. 9.

As can be seen in Table 11 and FIG. 9, the control group had no control effect, but the seed coated with the G341 strain showed 44% control effect. Therefore, it was confirmed that the seed coating with G341 strain can effectively control foot blight.

Example 14 Control of Tomato Root-knot Nematode Disease by Seed Coating

Seeds coated with the G341 strain and uncoated seeds were sown in a greenhouse, grown for one month, and then planted in a plastic house in Goesan, Chungcheongbuk-do. Four hundred 100-week diets were established and after 60 days the number of live and dead tomato plants was counted (Table 12). In the case of the soil where tomato foot blight was severely affected every year, the soil analysis confirmed that the density of root-knot nematodes is very high. By taking the plant roots withered and observing the nodules, it was confirmed that the root nodules were caused by M. incognita . Root-knot nematode control effect by seed coating using the G341 strain is shown in Table 12 below.

As can be seen in Table 12, there was no control effect in the non-treated control group, the seed coated with the G341 strain showed a control effect of 39%. Therefore, in combination with seed coating treatment, G341 strain and protease treatment at the time of treatment, and culture solution of G341 strain at the time of onset, effectively prevent complex plant diseases caused by tomato foot blight and root-knot nematode disease, and fusidium oxysporum and root-knot nematodes. It is expected to be able to control.

Example 15 Control Effect of Red Pepper Plague by Solid Cultures of G341 Strain

40 ml of the liquid culture prepared by incubating the G341 strain at 25 ° C. and 150 rpm for 24 hours in a BHI (Brain Heart Infusion) medium at 2 ° C. for 2 minutes at 121 ° C. : 13, w / w / w, 140 ml of DW), then 21.7 ml of glucose solution (0.45 g glucose / ml), 1.95 ml of 1 M KH ₂ PO ₄ solution and 3.9 ml of 1 M MgSO ₄ solution After addition, it was incubated for one week at 30 ℃. 20 ml of the prepared solid cultures were treated on the surface of pots (horticultural topsoil: 200 ml) of pepper seedlings of 6 leaf stages. After growing for one week in a greenhouse, the inoculator was inoculated with a fertilizer suspension of pepper blight. At this time, the pepper pest bacterium was obtained as follows. Specifically, after culturing for 1 week in pepper oatme ( Phytophthora capsici ) in the oatmeal agar medium, the aerial mycelium of the pathogen was removed and then light-treated at 25 ℃ for 24 hours. After harvesting the seedling sac with sterile distilled water at 4 ° C., the concentration was adjusted to 3 × 10 ³ seedling sac / ml. After one hour at 4 ° C. to release the strainer, 10 ml of the strainer was irrigated to the soil. Inoculated peppers were wet-treated in a wet room (relative humidity 95%, 25 ° C.) for 24 hours, transferred to a greenhouse, and bottomed with water. The experiment was repeated five times and the incidence was examined every day until 70% or more of the plants in the untreated group were shown in FIG. 10.

As shown in FIG. 10, in the case of the untreated group, the incidence gradually increased over time, and almost all plants withered and died after 19 days, whereas the solid culture treated group of the G341 strain was hardly developed, compared to the untreated group, 80 It showed a high control effect of%. Therefore, it was confirmed that the pepper blight can be effectively controlled when the G341 strain is treated by solid culture.

Example 16: Control of Chinese cabbage root-knot disease by solid culture of G341 strain

Chinese cabbage root gall disease (pathogen: Plasmodiophora) of a solid culture of G341 strain cultured in the same manner as in Example 15 The control effect on brassicae )) was investigated. Specifically, 5% of the solid culture of G341 strain was mixed with sterilized horticultural soil No. 5, and then seeded cabbage was staggered. Grown in a greenhouse for two weeks and transplanted into two soils. Lee Byeong-to was made by mixing finely cabbage root nodules from the pavement with a blender, gauze them, and inoculating the obtained solution in 1% of the soil. The experiment was repeated five times and was repeated in the same manner. Three weeks after the inoculation, the plants were pulled to remove the soil from the roots and the incidence was examined.

The incidence was defined as 0: sound, 1: root nodules formed in the roots, 2: root nodules formed in the main roots, 3: root nodules formed entirely in the roots and main roots, 4: large root nodules formed in the roots and main roots. , The average value is shown in the incidence degree, and the control value thereof is calculated and shown in Table 13 below.

Control Effects of G341 Strain Solid Cultures on Chinese Root Disease process Onset Control price (%) G341 Strain Solid Cultures 1.2 68% Non-treatment 3.7 -

As shown in Table 13, the solid culture of the G341 strain exhibited a 68% control against Chinese cabbage root-knot disease. Therefore, it was confirmed that when the G341 strain was treated with a solid culture, it was possible to effectively control Chinese cabbage root-knot disease.

Example 17 Effect of Controlling withered Diseases on Solid Cultures of G341 Strains

The control effect against the withering disease (pathogen: Fusarium oxysporum f. Sp. Raphani ) of G341 strain cultured in the same manner as in Example 15 was investigated.

Specifically, 8 x 16 plugs containing sterile horticultural soil No. 5 soil and soil mixed with sterile vermiculite 1: 1 (v / v) were seeded with susceptible varieties, Cheongsugung-mung and Jangsaeng-mu, which are medium-tolerance varieties. Cultivation was carried out for 14 days. Two weeks later, the roots were removed from the plug seedlings, washed with water, and soaked for 30 minutes in the suspension of the withered bacteria. At this time, the spore suspension was obtained as follows. Specifically, Huzium oxysporum form spysis raffini (Gangneung University) was inoculated in MEB (Malt extract broth) medium and cultured at 25 ° C. and 150 rpm for 7 days. After removing the mycelium, it was prepared by adjusting the spore concentration to 1 x 10 ⁷ conidia / ml concentration. The soaked seedlings were taken out and transplanted into 5 x 8 plugs using sterile horticultural tops, where solid cultures of the G341 strain were mixed at a 5% level. After wet treatment for 24 hours on the wet phase (95% relative humidity, 25 ℃) was transferred to the greenhouse to investigate the incidence after 3 weeks.

The incidence is 0: wholesome, 1: underground part browned, ground part not wilted and without symptoms, 2: underground part browned, ground part wilted, 3: underground part browned, ground part wilted and yellowed, 4: underground part It was defined as browned and ground part severely yellowed, withered and deciduous, 5: dead, and its average value was expressed as incidence, and its control value was shown in Table 14 below.

As shown in Table 14, the G341 strain solid culture showed control values of 65% and 80% in both varieties of umbilical uterus and Jangsaeng radish, respectively, and showed a high control effect against wilted rot compared to the control group.

Example 18 Control Effects on Tomato Foot Blight in Solid Cultures of G341 Strains

The control effect against tomato foot blight (pathogen: Ralstonia solanacearum ) of the solid culture of G341 strain cultured in the same manner as in Example 15 was investigated.

Specifically, after sowing in sterilized soil, tomato (cultivar: Seogwang) grown in a greenhouse for 3 weeks was transplanted into a pot containing sterilized horticultural soil. Solid pots of G341 strain in each pot were treated with 1% (2 ml), 5% (10 ml) and 10% (20 ml) of the horticultural soil contained in the pot and then grown in a greenhouse for 1 week. Harvested tomato foot blight bacteria shaken at 30 ° C. for 24 hours in CPG medium, adjusted to 10 ⁸ CFU / ml with sterile water, and then 20 ml of this suspension was irrigated in a pot (horticultural soil volume: 200 ml). It was. The wilting symptom was examined as incidence daily while maintaining a temperature of 30-35 ° C. after inoculation.

Incidence was defined as 0: no withered leaves, 1: 0-25% leaf wither, 2: 25-50% leaf wither, 3: 50-75% leaf wither, 4: 75-100% leaf wither. , The average value is shown in the incidence degree, and the control value thereof is calculated and shown in Table 15 below.

Control Effects of G341 Strain Solid Cultures on Tomato Foot Blight process Onset Control price (%) G341 Strain Solid Cultures 1.2 68 Non-treatment 3.8 -

As shown in Table 15, G341 strain solid culture showed a control value of 68%, and showed a high control effect against tomato foot blight compared to the control group.

Korea Institute of Bioscience and Biotechnology Biological Resource Center KCTC11497 20090406

Attach an electronic file to a sequence list

Claims (8)

Bacillus velezensis G341 strain (KCTC 11497BP) with antagonism against plant diseases. The microorganism preparation for plant disease control, containing any one or more selected from the group consisting of the Bacillus Velensis G341 strain of claim 1, the spores of the strain, the culture solution of the strain, and the extract of the culture solution. The method of claim 2,
The plant diseases include rice leaf blight blight, rice blight, tomato gray mold, tomato blight, tomato green blight, tomato wilting disease, tomato rootworm nematode disease, wheat rust disease, barley powdery mildew, pepper anthrax, pepper blight, lettuce fungus nucleus disease, ginseng dot pattern Microbial agent for plant disease control, characterized in that any one or more selected from the group consisting of deciduous disease, Chinese cabbage root gall disease, radish rot and cucumber root gall nematode disease. The method of claim 2,
The culture solution or the extract of the culture medium is characterized in that it comprises at least one active substance selected from the group consisting of Bacillomycin (Bacillomycin) L, pengycin (fengycin) A and subtilisin (subtilisin), for controlling plant diseases Microbial agents. The method of claim 2,
The extract of the culture solution is characterized in that the culture solution is extracted with ethyl acetate, butanol, water or a mixed solvent thereof, microbial preparation for plant disease control. A method for controlling plant diseases, comprising treating the plant or microorganisms for controlling plant diseases with a plant or the surrounding environment. The method according to claim 6,
The plant disease is rice leaf blight blight, rice blast, tomato gray mold, tomato blight, tomato green blight, tomato wilting disease, tomato root worm nematode disease, wheat red rust disease, barley powdery mildew, pepper anthrax, pepper blight, lettuce fungus nucleus disease, ginseng spot pattern It is any one or more selected from the group consisting of deciduous disease, Chinese cabbage root gall disease, radish rot and cucumber root gall nematode disease, the plant disease control method. The method according to claim 6,
The microbial agent is treated with soil, treated with plant surfaces, coated with seeds, or a combination thereof.

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