JPH03220108A - Novel microorganism - Google Patents

Abstract

PURPOSE:To obtain a novel microorganism belonging to Pseudomonas vandii, capable of increasing productivity of vegetable, etc., and readily controlling plant disease owing to plant pathogenic fungus of genus Fusarium in an excellent efficiency because of having an antifungal activity against said pathogenic fungus. CONSTITUTION:The objective novel microorganism is composed of Pseudomonas vandii VA-1316 (FERM P-10732), having an antifungal activity against plant pathogenic fungus belonging to genus Fusarium and able to readily control plant disease owing to said pathogenic fungus, especially soil borne disease such as putrefactive disease, chlorosis, dead arm, wilt disease, damping-off disease or dry rot disease in a high efficiency, then capable of strongly increasing productivity in a cultivation of vegetable, etc. Besides, said microorganism can be readily cultured as fluorishingly grows in either of a synthetic medium or a natural medium and able to stably produce various fermentation products in a high efficiency.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel microorganism, and more particularly to a microorganism having antibacterial activity against plant pathogenic bacteria of the genus Fusarium.

[Prior art and problems to be solved by the invention] Many plant diseases, such as leaf wilt and root rot, are known to be caused by plant pathogenic bacteria belonging to the genus Fusarium, and the damage caused by these causes a loss of income for farmers. is causing great damage.

The damage to highly profitable vegetables is particularly severe.

Continuous cultivation of highly profitable crops such as vegetables is currently difficult due to deterioration in the physical and chemical properties of soil and the occurrence of soil-borne diseases and soil pests.

Continuous crop failure is often caused by soil-borne diseases (most of these soil-borne diseases are caused by plant pathogenic bacteria that belong to the genus Fusarium. This is caused by the invading of plants, and there are few effective control agents, and conventional pesticides alone have not been able to prevent these plant diseases.

In addition, there are horticultural methods such as breeding of disease-resistant varieties and grafting to control these soil-borne diseases, but these methods require a long time, are not quick-acting, and are complicated. Not practical. Furthermore, among these soil-borne diseases, tomato wilt and strawberry yellow blight are examples of causing serious damage, but pesticides that can efficiently control these diseases are not yet known. .

Therefore, it has been desired to develop a new type of method for controlling soil diseases.

[Means for Solving the Problem] The present inventors have developed a method for controlling soil-borne diseases such as rot, yellowing, vine splitting, leaf wilt, damping-off, and dry rot. After a wide search for microorganisms that exhibit antibacterial activity against microorganisms belonging to the genus Fusarium, which are pathogens of plant diseases, we discovered a new microorganism that exhibits antibacterial activity against these soil-borne pathogens.

That is, the present invention is a novel microorganism that belongs to Pseudomonas bumpii and is characterized by having antibacterial activity against plant pathogenic bacteria of the genus Fusarium. A representative strain of this bacterium, Pseudomonas pandei (Pseud
The mycological properties of S. otmonas vandii) Shi A-1316 (Feikoken Bacteria No. 10732) are shown below.

(1) Cell shape and size: spherical or short culm-like radius 0.5-0.9 μm, length 0.5-1.5 μm + population, single or bicellular.

It is motile and has polar flagella.

Presence or absence of spores: Not produced.

Durham staining Durham negative.

Anti-acidity negative.

Extracellular adhesive is not produced.

Growth status in each of the following media (culture at 30°C for 3 days unless otherwise specified) (1)
Morphology and shape of colonies cultured on broth agar plates: External shape - round, size - 2-3 ml1, ridges - semicircular, structure - homogeneous 2 surface - smooth.

Margins - smooth and completely green Bicolor - white or yellowish-white.

Transparency - opaque, hardness - buttery.

(2) Meat juice agar slant medium Grows vigorously and uniformly along the inoculation line.

Ridges - Moderate 1 Surface - Smooth 9 Edges - Smooth.

(3) (4) (5) (6) (7) Color - white or yellowish white, transparency - opaque, hardness - buttery.

Broth liquid broth Grows all over. There is precipitation.

Does not form a closed ring.

peptone water liquid medium Grows vigorously throughout. There is precipitation.

Does not form a closed ring.

Meat juice agar puncture culture Grows uniformly in the shape of small papillae. It grows to a diameter of 2 to 4 squares on the surface of the medium.

Meat juice gelatin puncture culture Culture at 20°C for 10 days.

It grows vigorously throughout and liquefies gelatin.

lidmus milk culture Cultured at 30°C for 4 weeks.

Bacterial growth is observed and casein is digested, but no coagulation is observed.

3. Physiological properties (1) Reduction of nitrates: Reduce nitrates to nitrous acid.

(2) MR test Negative.

(3) VP test Negative.

(4) Indole production negative.

(5) Hydrogen sulfide generation Negative.

(6) Starch hydrolysis negative.

(7) Use of citric acid (combined use of Koser medium and Christensen medium).

(8) Utilization of nitrogen sources Ammonium hepta-salt, peptone and urea are respectively utilized as nitrogen sources. However, it does not utilize nitrates.

(9) Formation of pigment: No formation.

(10) Urease positive.

(11) Catalase positive.

(12) Generation of ammonia Generate.

(13) Range of growth It grows in the pH range of 4 to 8.

A pH range of 6 to 7.5 is preferred.

Grows at a temperature of 5-37°C. It does not grow at 42°C. 2
5 to 30°C is preferred.

(14) Attitude towards oxygen: aerobic.

(15) Oxidase reaction positive.

(16) O-F test (Hugh Lifeson Hugh
Based on the Leifson method. ) Decomposes sugar oxidatively, but not fermentatively.

(17) Assimilation of sugars, production of acid, and production of gas (see blank below) Assimilation: Assimilate and decompose.

+(w): Weakly assimilate or weakly produce acid.

(18) Assimilation of other carbon sources (19) Salt tolerance It does not grow in a medium containing 3% by weight NaCl.

(20) Vitamin requirement Absolutely does not require vitamins.

(21) Denitrification reaction Positive.

(22) CTC (guanine + cytosine) content 68.5
io1% (23) Main bacterial cell fatty acid composition Saturated fatty acids Unsaturated fatty acids (24) Main hydroxy acids 3-hydroxy acids 2-hydroxy acids (25) Quinone type C16:0 C16:L C18:1 30H-C14: 0 30H-C16:0 20) 1-C16:0 Ubiquinone Q @ (blank below) Explanation of abbreviations in (26) ATCC: American Type Culture Collection (A+merican Type Culture C
o11ection.

12031 Parklawn Drive Rock
ville.

MD 20852 USA) NTAES: Ministry of Agriculture, Forestry and Fisheries National Institute of Agricultural and Environmental Technology (Nati
onal In5titude of Agro-En
viron-mental 5science.

33-1-1 Kannondai, Tsukuba City, Ibaraki) JC: RIKEN Microbial System Preservation Facility (Japan)
Co11ection of Microorgan
iss+s.

2-1 Hirosawa, Wako City, Saitama Prefecture) T: Indicates a standard strain (Type 5 train).

(27) Isolation Source Soil The microorganism of the present invention (hereinafter referred to as the present bacterium) is a Durham-negative bacterium with polar flagella, and has a GC content of 68.5 a+o.
1%, and the main bacterial fatty acid composition is C16:O, C1
6:1. C18:1 and 3-hydroxy acid C1
4:0. C16:0 and 2-hydroxy acid C16
: O is the main hydroxy acid, and the quinone type is ubiquinone Q, so it is important to note that Virsey's Manual of Systematic Bacteriology (
Bergey's Manual of System
atic Bacteriology) Volume 1
Editors Krieg and Holt 0 (of
t): Williams Andu Wilkins (Will
Iams and Wilkins), (1984)
), it seems to be a strain belonging to rRN^ group H of the genus Pseudomonas. However, this bacterium and each species of rRNA group
They differ in denitrification reactions, acid production from sugar, and carbon source assimilation. Furthermore, each strain of rRNA group ■ is
They are clearly distinguished by DNA-DNA homology.

Therefore, the present inventors determined that this bacterium is a new bacterial species, and identified it as Pseudomonas bumpi (Pseu-doa+
onas vandi).

In the present invention, the method for investigating mycological properties is based on Bergey's Manual of Systematic Bacteriology.
f Systematic Bacteriology
) Volume 1 Editors: Cleve and Holt: Williams Andilkins (
Williams and'Wilkins), (
1984), ``Bacteriology Practice Summary'' (1958), edited by the Institute of Medical Science Alumni Association, and ``Classification and Identification of Microorganisms J'' (1975), edited by Takeharu Hase. The classification of this bacterium from soil was 0.025wt. It was carried out in a conventional manner using a broth medium (pH 7>) containing % fusaric acid.

The medium used for culturing this bacterium must contain a carbon source that can be assimilated by this bacterium, and may also contain an appropriate amount of nitrogen source, inorganic substances, etc., or a synthetic medium. or a natural medium.

The carbon source is not particularly limited as long as it can be assimilated by this bacterium, but natural products such as molasses and meat extract, D
- sugars such as glucose and D-fructose, D
- Sugar alcohols such as sorbitol and glycerol, organic acids such as succinic acid and acetic acid, alcohols such as ethanol, n-propyl alcohol, etc. can be used.

The concentration of these carbon sources in the culture medium varies depending on the type of carbon source, and is adjusted as appropriate to grow and multiply the microorganisms.
selected.

As nitrogen sources, use is made of inorganic nitrogen compounds such as, for example, ammonium salts and/or organic nitrogen-containing substances such as, for example, corn steep liquor, casein, peptone and meat extract.

In addition, examples of inorganic components include calcium salts, magnesium salts, potassium salts, sodium salts, phosphates,
Manganese salts, zinc salts, iron salts, copper salts, molybdenum salts, cobalt salts, boromine compounds, iodine compounds, and the like are used.

Furthermore, when using a strain that requires nutritional substances such as vitamins, the nutritional substances required by the strain are added.

The culture conditions include a temperature of 5 to 37°C, preferably 25 to 37°C.
30°C, and the pH is 4-8, preferably 6-7.
．． It is considered to be 5. Aerobic culture is performed under these conditions.

Although the growth and proliferation of this strain will be relatively poor, this does not preclude culturing under these conditions.

Further, the dissolved oxygen concentration of the culture solution is not particularly limited as long as it is a dissolved oxygen concentration that allows the present bacteria to grow and multiply, but it is usually preferably about 0.5 to 20 ppm+. In order to achieve such a dissolved oxygen concentration, there are various steps such as adjusting the amount of aeration gas, stirring, using oxygen gas or a gas mixture with air as the aeration gas, and increasing the pressure inside the culture tank. means are adopted.

Furthermore, the culture method may be batch culture, continuous culture, or semi-continuous culture.

, when ammonium salt is used as the nitrogen source,
During the culture period, ammonia is consumed for bacterial cell production and the pH of the culture solution decreases. In this case, the p
In order to maintain H at a predetermined value, it is preferred to add alkalis such as ammonia, caustic potash and caustic soda.

After culturing the present bacteria in this manner, the bacterial cells are separated from the culture solution by a conventional method. When the target substances are substances such as enzymes, coenzymes, and amino acids that are excreted outside the bacterial cells, these substances are extracted and separated from the culture solution and further purified. On the other hand, when the target substance is intracellular substances such as coenzymes, nucleic acids, vitamins, proteins, and lipids4, the bacterial cells are treated with various methods, the target substance is extracted and separated, and then the target substance is extracted and separated. , refined.

These obtained substances are effectively used as foods, medicines, feeds, feed additives, industrial raw materials, and the like.

In addition, this bacterium has antibacterial activity against plant pathogens belonging to the genus Fusarium, so it can be used to treat plants caused by pathogens belonging to the genus Fusarium such as sagebrush, tomatoes, strawberries, chives, radish, honeysuckle, burdock, asparagus, onions, cabbage, etc. cucumber, chisha, loofah, eggplant, melon, lotus,
Dry rot, plant blight, leaf wilt, damping-off, yellowing, vine splitting, root rot, which occurs in vegetables such as watermelon, radish, spinach, selto, chorogi, taro, yam, ginger, and udon. Plant diseases such as half blight and rot can be controlled.

In order to control these plant diseases, this bacterium or culture solution can be used as is, or since this bacterium excretes antibacterial substances outside the bacterial body, the culture supernatant liquid can be mixed with soil or applied to the soil surface. Alternatively, methods such as spraying the plant or immersing the plant in a culture solution or culture supernatant to attach the bacteria or the active ingredient can be employed.

Note that these methods require a large amount of bacteria because the bacteria used gradually die in nature.

Furthermore, when a culture supernatant is used, a large amount of culture supernatant is required because there are few antibacterial substances discharged into the culture solution.

For example, in order to efficiently control soil-borne plant diseases such as tomato wilt, it is necessary to inoculate plants that have an affinity for this bacterium, such as chives and green onions, with this inoculation. It is preferable to grow this bacterium in the soil by co-planting the plant with a plant that is the target of disease control (hereinafter referred to as the target plant), and to control the target pathogenic bacteria through the antibacterial action of this bacterium. .

[Examples] Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 1 g of soil was added to 1 d of sterile water aseptically and suspended. This suspension l- was poured into a meat broth medium (regular bouillon, Yoneken) containing 0.025 wt% fusaric acid, and heated at 28°C.
Static culture was performed at C for 7 days.

This culture solution was cultured on a gravy agar plate medium containing 0.025 wt% fusaric acid as described above, and a single colony that emerged was inoculated onto a gravy agar slant medium and cultured. From this, a pure strain of Pseudomonas vampii VA-1316 was obtained.

Pour 200 g of broth liquid medium into an 11-volume Erlenmeyer flask, and add this to the above-mentioned Pseudomonas bumpii VA-1316.
The absorbance (610 nm) of the culture solution obtained by inoculating and culturing at 30''C for 1 day was 4.0.

Usage example 1 Pseudomonas bandii VA-1 was placed on the surface of a potato dextrose agar medium (manufactured by Hinaga Pharmaceutical, composition: 20% potato extract, 2% glucose, 1.5% agar) in a petri dish.
316 and Fusarium oxysporum F.S. cucumolinum (a plant pathogenic fungus) were inoculated opposite each other at an interval of 3 cm, cultured at 25°C for 7 days, and the state of growth of the pathogenic fungus was observed.

As a result, the growth of pathogenic bacteria was inhibited.

Use Example 2 Pseudomonas vampii VA-1316 was cultured in a broth medium (manufactured by Difico) at 30''C for one day, and chive scales were immersed and inoculated into this culture solution.

The inoculated leek bulbs were mixedly planted at about 20 cm from the base of the tomato plants at the time of planting the tomatoes.

Table 1 shows the results of the control test for tomato root rot wilt.

Table 1 [Effects of the Invention] The bacteria of the present invention grow vigorously in both synthetic and natural media, can be easily cultured, and can efficiently and stably produce various fermentation products. It can be manufactured.

Furthermore, since this bacterium exhibits antibacterial activity against plant pathogenic bacteria belonging to the genus Fusarium, it becomes possible to efficiently and easily control plant diseases caused by plant pathogenic bacteria belonging to the genus Fusarium, and to cultivate vegetables, etc. This makes it possible to significantly improve productivity.

teenager Reason Man

Claims (1)

[Claims] A novel microorganism that belongs to Pseudomonas bandii and is characterized by having antibacterial activity against plant pathogenic bacteria of the genus Fusarium.