JP2010081837A - Burkholderia bacterium, plant disease-controlling agent and controlling method using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new microorganism which controls the growth of more kinds of plant-pathogenic microbes than those by conventional microorganisms and exhibits excellent control effects against plant diseases, has a degradation ability for widely degrading many kinds of replant-obstructing substances, and has also an excellent control action against replant failures: to provide a plant disease-controlling agent: and to provide a plant disease-controlling method which each uses the new microorganism, is high in safety, and little pollutes environments. <P>SOLUTION: There are provided the Burkholderia bacterium which controls plant diseases and degrades replant-obstructing substances; and the Burkholderia bacterium, in which the base sequence of 16S liposome DNA (16S rDNA) is a base sequence represented by sequence No. 1, and which exhibits a β-galactosidase activity. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a bacterium belonging to the genus Burkholderia (genus Burkholderia) having an ability to control a fungal plant disease that infects a plant, and an ability to control a continuous cropping disorder caused by benzoic acid and its derivatives, and a plant disease using the same The present invention relates to a control agent and a control method.

  In agricultural production, pest control is the most important. Chemical pesticides are used for the purpose of controlling pests, saving labor, stabilizing the quality and yield, etc. It is indispensable for current agriculture.

  On the other hand, chemical pesticides are highly toxic to humans from the viewpoint of insecticidal and sterilizing applications, and have a risk of adversely affecting the health of agricultural producers and consumers.

  The types and amounts of chemical pesticides that can be used are restricted from the viewpoint of safety according to the chemical pesticide use standards stipulated in the Agricultural Chemicals Control Law. In fact, there are many things that are concerned about chronic toxicity, carcinogenicity, teratogenicity, multi-generation genotoxicity, and the like.

  Recently, chemical pesticides are generally sprayed to prevent pathogen infection on crops. However, chemical pesticide residues on agricultural products are also a major problem, and the effects of chemical pesticides remaining on agricultural products on the human body There are many safety problems such as environmental pollution.

  Methods that use wood vinegar, bamboo vinegar, baking soda, electrolytic acid water, etc. in place of conventional chemical pesticides are known as safe and environmentally friendly methods for controlling plant diseases. Therefore, there is a need for a method for controlling plant diseases that is not sufficient in terms of safety and that is safe, has a high control effect, and can be carried out inexpensively.

  As one of them, plant disease control agents that contain microorganisms and use antagonism of microorganisms against plant pathogens and plant disease control methods using the same have been developed, some of which are microbial pesticides (plant disease control agents) ) Has been put into practical use.

  Examples of plant disease control agents using microorganisms and plant disease control methods using the same include control of strawberry anthracnose with Talaromyces flavus and ketium aureum. (Patent Document 1) and examples of controlling various plant diseases using Bacillus subtilis, which is a kind of bacteria, are known (Patent Document 2).

  On the other hand, continuous cropping failure in agricultural production is also a serious problem that lowers the productivity of crops. This is caused by the accumulation of growth inhibitors (successive cropping barriers) secreted from crop roots in soil, The increase in the density of phytopathogenic fungi, the increase in the density of nematodes in the soil, the deterioration of the physical properties of the soil, etc. are considered.

Continuous crop damage caused by phenol derivatives such as benzoic acid has been reported in many crops such as rice, strawberries, tomatoes, watermelons, cucumbers, lettuce, asparagus and taro.
Japanese Patent Laid-Open No. 10-229872 JP-A-5-51305

  By the way, the invention using ketium described in Patent Document 1 and the invention using Bacillus subtilis described in Patent Document 2 broadly suppress the growth of various types of phytopathogenic fungi (having a wide action spectrum). Moreover, it is not described whether it has the effect of decomposing the continuous cropping obstacle substance contained in the soil.

  The object of the present invention is to suppress the growth of more types of phytopathogenic fungi than before, to exhibit excellent control effects against plant diseases, and to have the ability to decompose a wide variety of continuous crop disorder substances. To provide a new microorganism having an excellent control action against continuous cropping disorders, and also to provide a plant disease control agent and a control method with high safety and low environmental pollution by using the new microorganism. It is in.

  The present invention is a bacterium belonging to the genus Burkholderia which controls plant diseases and decomposes substances that hinder continuous cropping.

  As a result of intensive research to solve the above problems, the present inventors have succeeded in isolating a novel microbial strain belonging to the genus Burkholderia (genus Burkholderia) that can exert an excellent effect on plant disease control. did.

  Furthermore, as a result of investigating the growth inhibitory effect against various plant pathogens using this strain, spinach blight fungus (Pythium. Sp.), Rice blight fungus (Fusarium, Pythium, Rhizopu, Trichoderma), spinach dwarf fungus (Fusarium oxysporum) sp. spinaciae), Fusarium oxysporum f. sp. conglutinans Cong, Fusarium oxysporum f. sp. fragariae, Fusarium oxysporum f. sp. lycopersici and f. sp. Inhibitory activity against radicis-lycopersici), radish-oxysporum f. Found to show.

  In addition, as a result of investigating the degradation ability of phenol derivatives (substance cropping disorder substances) that have been reported to be involved in continuous cropping disorders, benzoic acid, salicylic acid, m-hydroxybenzoic acid, p-hydroxybenzoic acid, protocatechuic acid, vanillin It has been found that acids, syringic acid, gentisic acid and 2,4-dichlorobenzoic acid all have a broad decomposition spectrum.

  Furthermore, it discovered that it showed the control effect using various plant disease generation | occurrence | production system and continuous cropping failure generation | occurrence | production system, and came to complete this invention.

  The microorganism of the present invention belongs to the genus Burkholderia and does not show pathogenicity to animals and plants. Preferably, the Burkholderia genus (Burkholderia genus) CRSE-3 strain isolated from the strawberry root by the inventor is mentioned.

  The CRSE-3 strain is a novel isolate, and as described above, has an extremely excellent property that it has the ability to degrade a phenol derivative that has been reported to inhibit the growth of phytopathogenic bacteria and to be involved in continuous cropping disorders. is doing.

  By providing new microorganisms that suppress the growth of various types of plant diseases and decompose many types of continuous cropping obstacles, they have excellent control effects such as having a broad spectrum of action against plant diseases of crops. Demonstrates and can prevent crop failures caused by continuous cropping substances.

  Furthermore, since the new microorganism is harmless to humans, the use of this new microorganism can provide a plant disease control agent that is highly safe and has little environmental pollution.

  As a result of searching for microorganisms capable of degrading various phenol derivatives that have been reported to exhibit growth inhibitory effects against various plant pathogens and to be involved in continuous cropping disorders, the inventor of Takamatsu City, Kagawa Pref. We succeeded in isolating Burkholderia genus CRSE-3 from strawberry roots cultivated at Shikoku Research Institute.

  The bacteriological properties of this strain are as follows.

  The CRSE-3 strain can be grown by aerobic culture at Nutrient agar plate at 30 ° C. for 1 to 3 days. As a morphological feature, there is no spore formation, and the above culture day has a diameter of 1 mm. , Pale yellow, round, lenticular, smooth, opaque, butter-like colonies are formed.

  The preferred culture temperature of the CRSE-3 strain is 25 ° C. to 37 ° C., and its cell form is a gonococcus having a size of about 0.7 to 0.8 × 1.2 to 1.5 μm, has motility, and flagella staining shows polar flagella. Yes, Gram stain:-(negative).

  The physiological properties of the CRSE-3 strain are catalase: +, oxidase:-, acid / gas production (glucose):-/-, O / F test (glucose):-/-(+: positive,-: negative) It is.

  The utilization of the CRSE-3 strain is as follows: D-glucose: +, L-arabinose: +, D-mannose: +, D-mannitol: +, N-acetyl-D-glucosamine: +, maltose:-, trehalose: -, Sucrose:-, L-arginine: +, potassium gluconate: +, n-capric acid: +, adipic acid: +, DL-malic acid: +, sodium citrate: +, phenyl acetate: + (+: Assimilate,-: not assimilate).

  The CRSE-3 strain reduces nitrate, does not hydrolyze esculin (no β-glucosidase activity), exhibits β-galactosidase activity, and grows in the presence of 1.5% NaCl. In particular, it differs from the properties of Burkholderia fungorum in that it exhibits β-galactosidase activity.

  The 16S rDNA base sequence of the CRSE-3 strain is as shown in the base sequence 1 (SEQ ID NO: 1 in the Sequence Listing, see FIG. 6), and BLAST (pubMed (http: / as a result of homology search against the bacterial reference strain database using the No. AF215705) 16S rDNA (SEQ ID NO: 2 in the sequence listing, see FIG. 6), the nucleotide sequences at three positions differed, indicating 99.8% homology.

  From the above properties, it was confirmed that the CRSE-3 strain is a new species of Burkholderia genus that is very closely related to Burkholderia fungorum.

  The CRSE-3 strain is deposited as a Burkholderia bacterium CRSE-3 at the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation, the deposit number is NITE P-486.

  For culturing the CRSE-3 strain, a normal bacterial culture method can be used, and a shaking culture method using a liquid medium or an aeration and agitation culture method is preferred.

  As the carbon source of the nutrient medium to be used, for example, carbohydrates such as glucose, fructose and sucrose are preferable, and as the nitrogen source, for example, casein decomposition products such as peptone, polypectone, and bacttotryptone, meat extract, soybean Boiled enzyme degradation products and the like are preferred, but are not necessarily limited thereto.

  The CRSE-3 strain is different from the conventional Burkholderia fungorum in that the nucleotide sequence at three positions in 16S rDNA is different and that it also exhibits β-galactosidase activity. A molecular phylogenetic tree of CRSE-3 strain based on 16S rDNA is shown in FIG.

  Previous literature on Burkholderia fungorum (Coenye T, Laevens S, Willems A, Ohlen M, Hannant W, Govan JR, Gillis M, Falsen E and Vandamme P., Burkholderia fungorum sp. Nov. And Burkholderia caledonica sp. Nov., Two New species isolated from the environment, animals and human clinical samples., Int. J. Syst. Evol. Microbiol., 51, 1099-1107 (2001)), CRSE-3 strain is different from Burkholderia fungorum A new species of Burkholderia sp.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, the scope of the present invention is not limited to these Examples. In addition, procedures that require aseptic operation are assumed to be aseptic including those described.

<Isolation of bacteria>
A method for isolating the Burkholderia bacterium CRSE-3 strain will be described.

  Strawberries (variety 'Smile Ruby' grown at Shikoku Research Institute. Registered No. 6562 of the Ministry of Agriculture and Fisheries) were cultivated in the same pot for more than a year, and aging roots were used as an isolation source.

  The roots were collected, shaken in 70% (v / v) ethanol for 1 minute, then shaken in a 1,000-fold diluted sodium hypochlorite solution for 2 minutes, and attached to the roots. Microorganisms were sterilized.

  As the liquid medium, a liquid medium containing benzoic acid as a sole carbon source (benzoic acid medium, see Table 1) was used in order to facilitate isolation of benzoic acid-utilizing bacteria living in the roots. 300 ml of a liquid medium was added to an Erlenmeyer flask (500 ml volume), stoppered with a breathable silicon stopper, sterilized by autoclave and used for culture.

  The sterilized strawberry root was washed 5 times with sterilized distilled water, 0.3 g of which was added to the Erlenmeyer flask culture solution, and then shaken at 30 ° C. for 3 days. Concentrated culture was performed.

  The resulting enriched culture was streaked on a benzoic acid medium containing 1.5% (w / v) agar to isolate benzoic acid-utilizing bacteria. The benzoic acid medium was the same as the liquid medium except for the agar composition.

  Benzoic acid-assimilating bacteria obtained by colony isolation were cultured for 5 days at 30 ° C. on strawberry anthrax (Glomerella cingulata NBRC 6426) and potato dextrose agar medium (PDA medium).

  Observation was carried out after culturing the cells against each other, and benzoic acid-assimilating bacteria having the ability to suppress the growth of strawberry anthracnose bacteria were selected as antagonistic bacteria.

  As a result of narrowing down the strains having the highest ability to suppress the growth of strawberry anthracnose fungi from the selected antagonistic bacteria, the CRSE-3 strain was successfully isolated.

＜安全性試験＞
単離したＣＲＳＥ−３株が動植物に対し毒性、病原性がないことを確認するために、ラットおよび各種農作物に対するＣＲＳＥ−３株の影響を調べた。
＜ラットによる安全性確認試験＞
ラット（系統：Crl:CD(SD)、微生物レベル：SPF、週齢：5週齢）雌雄各5匹を用意し、ラット1匹あたり1.0 ×１０⁸cfu (colony forming unit)のＣＲＳＥ−３株の菌体を経口投与した。

<Safety test>
In order to confirm that the isolated CRSE-3 strain is not toxic or pathogenic to animals and plants, the influence of the CRSE-3 strain on rats and various crops was examined.
<Safety confirmation test using rats>
Prepare 5 males and 5 females (strain: Crl: CD (SD), microorganism level: SPF, week age: 5 weeks old), and CRSE-3 strain of 1.0 × 10 ⁸ cfu (colony forming unit) per rat Were orally administered.

  After oral administration, the rats were raised for 21 days under the conditions of temperature: 19.0-25.0 ° C., humidity: 35.0-75.0%, lighting time: 12 hours / day.

  As animal feed, experimental animal solid feed (MF, manufactured by Oriental Yeast Co., Ltd.) was used. Rats were allowed to eat ad libitum except for the fasting period from the evening before the oral administration of the cells of the CRSE-3 strain to about 3 hours after administration on the day of administration.

  During the breeding period of rats, observation of life and death of each rat and body weight measurement were performed. All rats 21 days after oral administration were subjected to intraperitoneal administration of pentobarbital sodium (Nembutal, manufactured by Dainippon Pharmaceutical Co., Ltd.), and the abdominal aorta was cut and exsanguinated and euthanized. Thereafter, macroscopic examination of rats and pathological examination of major organs were performed.

As a result, no death, shortening of life span, and pathological change of rats considered to be caused by administration of the CRSE-3 strain were observed.
<Influence on crops>
The influence of the CRSE-3 strain on each crop (strawberry, tomato, komatsuna, rice) was examined.

First, the CRSE-3 strain was inoculated into 10 ml of Trypticase Soy Broth (TSB) medium (see Table 2) in an L-shaped test tube and cultured with shaking at 30 ° C. for 2 days. Thereafter, the cells obtained by centrifuging the culture solution were washed three times with sterilized water and adjusted to 1 × 10 ⁸ cfu / ml to obtain a cell suspension. This bacterial cell suspension was sprayed once a week on strawberries, tomatoes, komatsuna, and rice until the entire plant body, the leaf surface, and the back surface.

  As a result, no pathogenicity of the CRSE-3 strain to each crop was observed.

＜試験１＞
単離したＣＲＳＥ−３株の各種植物病原菌に対する増殖抑制作用を調べる試験を行った。

<Test 1>
A test was conducted to examine the growth inhibitory action of the isolated CRSE-3 strain on various plant pathogens.

As shown in Table 3, the plant pathogens used in this growth inhibition test were spinach blight ( _{Pythium ultimum var. Ultimum NBRC 32426} ), rice blight ( _{Pythium spinosum NBRC 32423} ), spinach wilt ( _{Fusarium) oxysporum sp. Spinaciae NBRC 30467} ), cabbage _dwarf fungus ( _{Fusarium oxysporum sp. Conglutinans NBRC 9469} ), strawberry _dwarf fungus ( _{Fusarium oxysporum sp. fragariae NBRC 31180} ), tomato _dwarf fungus ( _{Fusarium oxysporum sp. NB Lycopersic} ) Radish yellow rot ( _{Fusarium oxysporum sp. Raphani NBRC 9972 strain} ), tomato half body wilt fungus ( _{Verticillium dahliae NBRC 9939 strain} ), strawberry anthracnose fungus ( _{Glomerella cingulata NBRC6425 strain} ), strawberry leaf blight anthracnose fungus ( _{Colletotrichum orbiculare NBRC 33 strain} ) It is a gray mold fungus ( _{Botrytis cinerea NBRC 9760 strain} ).

  First, a plurality of PDA media (manufactured by Nissui Pharmaceutical Co., Ltd.) were prepared, and plant pathogens were inoculated by type in the center of each PDA medium. However, since the tomato half-wilt disease was slow to grow, it was pre-cultured for 2 days in advance at 25 ° C. in the dark, and the bacterial flora was used for inoculation.

After inoculating each phytopathogenic fungus, add CRSE to the part of the same medium about 3 cm away from the inoculated part.
-3 strains were inoculated in the range of 1 cm × 1 cm.

  After culturing the inoculated plant pathogen and the CRSE-3 strain at 25 ° C. under dark conditions for 3 days, observe and measure the formation of a growth-inhibiting zone against each plant pathogen and examine the growth of the plant pathogen by examining the growth of the plant pathogen Were evaluated for their ability to antagonize each plant pathogen.

  Evaluation criteria are +++: extremely strong antagonism (phytopathogenic growth almost stops), ++: strong antagonism (with a clear inhibition zone of 1 mm or more), +: antagonism (inhibition of less than 1 mm) There is a band),-: No antagonistic ability.

  The results are shown in Table 3. As can be seen from Table 3, the growth of the above-mentioned phytopathogenic fungi other than the gray mold fungus is suppressed by the CRSE-3 strain, and the CRSE-3 strain acts on a wide variety of phytopathogenic fungi to suppress its growth. It could be confirmed.

  This indicates the possibility that the CRSE-3 strain can suppress not only strawberry anthracnose but also various plant diseases.

＜ＣＲＳＥ−３株による生育阻害物質の分解試験＞
単離したＣＲＳＥ−３株について、代表的な生育阻害物質の分解能を調べた。

<Degradation test of growth inhibitory substance by CRSE-3 strain>
For the isolated CRSE-3 strain, the resolution of representative growth inhibitors was examined.

The CRSE-3 strain was inoculated into 10 ml of Trypticase Soy Broth (TSB) medium (see Table 2) in two L-shaped test tubes and cultured overnight at 30 ° C. with shaking. As a result, the bacterial concentration of the culture solution was about 0.731 and 0.6216 in absorbance (OD ₆₀₀ ) at a wavelength of 600 nm.

  The bacterial concentration in the preculture was measured using TAITEC PHOTO METER mini photo 518R (manufactured by Taitec Co., Ltd.) (hereinafter, the same instrument was used for measuring the absorbance).

  Prepare a plurality of test tubes containing 10 ml of W medium (aromatic compound metabolism medium, see Table 4). Benzoic acid, salicylic acid, m-hydroxybenzoic acid, p-hydroxybenzoic acid, protocatechuic acid, vanillic acid, syringic acid, A substrate solution of gentisic acid and 2,4-dichlorobenzoic acid was prepared, and the substrate solution was added to each test tube so that one type of substrate was contained in one W medium at a concentration of 20 mM.

Ｗ培地に前培養液200μlを添加して27℃、125rpmの条件で振とう培養を行った。培養開始から3日後に培養液の600nmにおける吸光度を測定してＣＲＳＥ−３株の増殖を確認した。

A preculture solution (200 μl) was added to W medium, and shaking culture was performed at 27 ° C. and 125 rpm. Three days after the start of culture, the absorbance of the culture solution at 600 nm was measured to confirm the growth of the CRSE-3 strain.

  The results are shown in Table 5. As can be seen from Table 5, it was confirmed that the CRSE-3 strain can be decomposed and utilized (assimilated) using almost all growth-inhibiting substances as nutrient sources. In particular, 2,4-dichlorobenzoic acid which is a hardly-degradable chlorine compound Was confirmed to be assimilated to some extent.

The disease control effect of CRSE-3 strain on spinach dwarf disease was confirmed by a test by seed cultivation.
Peat moss which was autoclaved in 128 holes seedling tray filled soil (organic carrier) consisting mainly of, 1 × 10 ⁸ cfu / ml bacterial suspension CRSE-3 strain was adjusted to the respective holes of 128 holes seedling tray 10 ml aliquots were dispensed.
Thereafter, 15 seeds of spinach 'Mirei' (manufactured by Sanyo Seed) were sown in each hole and cultivated in an artificial weather chamber adjusted to a temperature of 25 ° C, a humidity of 80%, an illuminance of 20,000 Lux, and a day length of 12 hours.
Seven days after sowing, 5 ml each of conidial suspension of Fusarium oxysporum f. Sp. Spinaciae (NBRC No. 30467) adjusted to 2.3 × 10 ⁵ cfu / ml was dispensed and inoculated. .
A group in which only CREW-3 strain was not added and inoculated with only yellow wilt bacteria was designated as control group A, and a group in which neither CRSE-3 strain was added nor yellow mold was inoculated was designated as control group B.
Seven days after inoculation with the yellow-yellowing fungus, germination rate and survival rate (ratio of dark green individuals in cotyledons and true leaves) were investigated.
As a result, as shown in FIG. 1, in the control group A, no symptom was observed on the leaves and stems of the cultivated plants, and the survival rate was 80%. In the control group B, cotyledon yellowing due to yellowing progressed, and the survival rate was about 70%.

  On the other hand, in the test group 1, the proportion of individuals whose cotyledons are yellowed due to yellowing is reduced, and the survival rate of the cultivated plants is improved by 10% compared to the control group A and 20% compared to the control group B. Addition of CRSE-3 strain showed high disease control effect against spinach wilt.

The disease control effect of the CRSE-3 strain on the blight of Chingensai was confirmed by a test by seed cultivation.
A 128-well seedling tray was filled with soil containing mainly peat moss sterilized by autoclaving, and 10 ml of CRSE-3 strain adjusted to 1 × 10 ⁸ cfu / ml was dispensed into each hole.
The above soil may contain an inorganic porous carrier (inorganic carrier) such as zeolite, attapulgite, sepialite, montmorillonite, pearlite, Kanuma soil, Akadama soil, pumice, charcoal, coral sand and the like.
After that, seeds of Chingensai 'Changyang' (manufactured by Takii Seed Co., Ltd.) were sown in each hole by 15 seeds and cultivated for 7 days in an artificial weather chamber adjusted to a temperature of 25 ° C, humidity of 80%, illuminance of 20,000Lux, and 12-hour day length This plant was used for the test.
Bacterial fungus (Pythium ultimum Trow var. Ultimum, NBRC No.32426) was cultured at 25 ° C. in a PDA medium (manufactured by Nissui Pharmaceutical Co., Ltd.) in a petri dish, and the bacterial flora of the fungus after culturing was sterilized The medium was cut into φ5 mm with a cork borer, etc., inoculated into a 500 ml Erlenmeyer flask containing 200 ml of potato dextrose (PD) liquid medium obtained by removing agar from PDA medium, and cultured with shaking at 25 ° C. for 7 days. After culturing, the culture solution was diluted 10-fold with sterilized water, and 5 ml was inoculated per well of the tray.
Twelve days after the inoculation, the number of germinated stalks in the middle of the 16 holes was investigated. A group in which neither the CRSE-3 strain was added nor inoculated with the blight fungus was designated as a control group C, and a group in which no CRSE-3 strain was added and only the blight fungus was inoculated was designated as a control group D.

  As a result, as shown in FIG. 2, in the control plot C, no symptom was confirmed on the leaves and stems of the cultivated plants, and the survival rate was 80%. In control group D, cotyledon yellowing due to yellowing progressed, and the survival rate was about 70%.

  On the other hand, in Test Zone 2, by adding the CRSE-3 strain to the medium, the proportion of cotyledonized individuals due to the bacterial wilt decreased, and for the survival rate, the addition of the CRSE-3 strain was also inoculated with the yellow rot fungus. It was about 10% better than the target group C, which was not performed, and about 23% better than the control group D, showing a disease control effect similar to the disease control effect on spinach dwarf disease.

The inhibitory effect of CRSE-3 strain on strawberry anthracnose by foliar application was investigated.
The entire leaves of strawberry seedlings (3 developed leaves) were inoculated by uniformly spraying the CRSE-3 strain suspension adjusted to 1 × 10 ⁸ cfu / ml with a hand spray.
After spraying the CRSE-3 strain, the strawberry seedlings were carried into a climate chamber controlled at a temperature of 20 ° C., a humidity of 80%, an illuminance of 20,000 Lux, and a day length of 12 hours.
One day after spraying the CRES-3 strain, a conidial suspension of strawberry anthracnose fungus (Glomerella cingulata NBRC6425 strain) adjusted to a concentration of about 10 ⁶ cells / ml is spread evenly over the strawberry seedlings by hand spraying. The strawberry seedlings were inoculated with anthracnose fungi.
After inoculation with anthracnose fungi, strawberry seedlings were covered with a plastic bag and cured for 2 days under conditions of room temperature 20 ° C, humidity 90%, illuminance: 20,000 Lux, day length 12 hours.
After curing, the plastic bag was removed, and cultivation was carried out for 2 weeks in an artificial weather room with a room temperature of 30 ° C, humidity of 85%, illuminance of 20,000 Lux, and a day length of 12 hours.
Two weeks after the start of cultivation, strawberry seedlings were observed, and the strains that developed lesions on the leaves of the strawberries were determined as diseased strains, and the strains in which they were not confirmed were determined as healthy strains. That is, a strain in which no lesions were found on the leaves of strawberries was regarded as a healthy strain, and a strain in which lesions were found on the leaves was designated as a diseased strain. Further, the diseased strains were evaluated in three stages according to the degree of lesions (slight lesions, clear lesions, and significant lesions).

  As a result, as shown in FIG. 3, in the control group E, the disease incidence was high, but in the test group 3 sprayed with the CRSE-3 strain, the degree of disease was reduced.

  It was investigated by hydroponics how much the reduction effect can be obtained by providing CRSE-3 strain against the growth failure of crops caused by benzoic acid, which is a typical continuous cropping obstacle substance.

  This fertilizer nutrient solution is a 1 / 2-concentration garden prescription nutrient solution (see Yoshiya Yamazaki, “Nutrient Solution Cultivation (revised edition), Hirotosha, published in 1987, p. 107)”. After adding benzoic acid to 2 mM, filter with a membrane filter made of polyethersulfone (PES) with a pore size of 0.22 μm (PES Bottle Top Filter 150 ml, 500 ml, manufactured by Nalge Nunc International Corp.) ) Sterilized.

  Dispense 200 ml of this fertilizer nutrient solution into a 500 ml Erlenmeyer flask, inoculate the fertilizer nutrient solution with CRSE-3 strain (hydroponic solution treatment), and plug a breathable silicone stopper for 7 days at 25 ° C and 120 rpm. Cultured with shaking. Silicone stoppers and Erlenmeyer flasks were sterilized in advance.

The fertilizer nutrient solution after the culture contained the CRSE-3 strain at a density of about 1.3 × 10 ⁹ cfu / ml. The cultured fertilizer nutrient solution was added to a plastic container with a lid of 370 ml (plant box, manufactured by Asahi Techno Glass Co., Ltd.), and the rooting was immersed in the fertilizer solution.

  Thereafter, this seedling seedling seedling was cultivated in an artificial weather chamber controlled at a temperature of 25 ° C., a humidity of 80%, an illuminance of 20,000 Lux, and a day length of 12 hours.

  In addition, the section cultivated with the fertilizer nutrient solution to which neither benzoic acid nor the CRSE-3 strain was added was the control plot F, and the plot cultivated with the fertilizer nutrient solution to which only the benzoic acid was added and the CRSE-3 strain was not added was the control plot. G.

  The survey was conducted on the seventh day of cultivation, and the main root length, hypocotyl length, cotyledon length, longest side root length, and number of side roots were examined.

  As a result, as shown in FIG. 4, vigorous growth was observed in the control group F in which neither benzoic acid nor the CRSE-3 strain was added, and the control group G in which only benzoic acid was added but no CRSE-3 strain was added. The growth was significantly reduced.

  On the other hand, in the test group 4 to which benzoic acid and the CRSE-3 strain were added, the growth (hypocotyl length, cotyledon length, number of lateral roots) was almost the same as the control group F to which nothing was added. It was shown that the CRSE-3 strain has high resolution against continuous cropping disorder substances.

  It was investigated how much the growth damage caused by benzoic acid was reduced by adding the CRSE-3 strain to the soil (soil treatment).

Fill the 128-hole seedling tray with soil containing mainly peat moss sterilized by autoclaving, add 10 ml of CRSE-3 strain adjusted to 1 × 10 ⁸ cfu / ml to each hole, and seeds of Chinggensai 'Changyang' into each hole 15 seeds (Takii Seedling) were sown.

  For irrigation, a 1 / 2-concentration garden prescription nutrient solution containing benzoic acid at a concentration of 0 mM, 2 mM, 10 mM, and 20 mM is supplied from the bottom of the 128-hole seedling tray, temperature 25 ° C, humidity 80%, illuminance 20,000 Lux Cultivated in a climate chamber adjusted to 12 hours of day length.

  Moreover, what added benzoic acid by 0 mM, 2 mM, 10 mM, and 20 mM density | concentration to the 1/2 density | concentration garden prescription nutrient solution without adding CRSE-3 strain | stump | stock was used as control group HK.

  The survey was conducted on the seventh day of cultivation, and the germination rate and cotyledon length were measured.

  As a result, as shown in FIG. 5, when all the seedlings that were sown were grown healthy and the cotyledon length was 10 mm or more, and the healthy growth rate was 100%, the control group H without benzoic acid added (0 mM) The healthy growth rate was 86.4%, but the healthy growth rate dropped to 24.4% in the control plot K containing 20 mM benzoic acid.

  On the other hand, in the test group 5 in which the CRSE-3 strain was added to the culture medium, the healthy growth rate recovered to 60.6% even in the test group 8 in which the growth was recovered and 20 mM benzoic acid was added. The three strains have high resolution even against continuous cropping substances contained in soil and soil, as shown in the hydroponic culture test of Example 5, and as a result, the growth of Chingen rhinoceros is restored. It has been shown.

It is a figure which shows the spinach dwarf yellowing effect of CRSE-3. It is a figure which shows the Chingensai blight reduction effect of CRSE-3 strain | stump | stock. It is a figure which shows the strawberry anthracnose reduction effect of CRSE-3 stock | strain. It is a figure which shows the growth inhibitory reduction effect by the benzoic acid of CRSE-3 strain | stump | stock by hydroponics. It is a figure which shows the growth inhibition reduction effect by the benzoic acid of CRSE-3 stock addition culture. The base sequences of rDNA of CRSE-3 strain and B. fungorum are shown. The molecular phylogenetic tree of CRSE-3 strain based on the base sequence of 16s rDNA is shown. The number near the branch of the branch indicates the bootstrap value, and the lower left line indicates the scale bar. The T at the end of the strain name indicates that type strain.

SEQ ID NO: 1 rDNA (1472mer.) Of Burkholderia sp. (CRSE-3 strain)
Sequence number 2: Burkholderia fungorum rDNA (1472mer.)

Claims (8)

  A bacterium belonging to the genus Burkholderia that controls plant diseases and decomposes substances that hinder continuous cropping.   The Burkholderia bacterium according to claim 1, wherein the base sequence of 16S ribosomal DNA (16S rDNA) is the base sequence shown in SEQ ID NO: 1 and exhibits β-galactosidase activity.   The Burkholderia bacterium according to claim 1, which is a CRSE-3 strain (NITE P-486).   A plant disease control agent comprising the Burkholderia bacterium according to any one of claims 1 to 3.   A plant disease control agent comprising an organic carrier or an inorganic carrier to which the Burkholderia bacterium according to any one of claims 1 to 3 is adsorbed.   A disease control method, wherein the soil treatment or hydroponic solution treatment is performed using the Burkholderia bacterium according to any one of claims 1 to 3.   A disease control method comprising spraying a cultured cell suspension of Burkholderia bacteria according to any one of claims 1 to 3 to a plant body.   A disease control method comprising immersing the root of a plant body in a cultured cell suspension of a Burkholderia bacterium according to any one of claims 1 to 3.

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