JPS63198974A - Microorganism having ability to decompose fusaric acid and decomposition of fusaric acid using said microorganism - Google Patents

Abstract

PURPOSE:To decompose and inactivate fusaric acid, by an applying a novel microorganism, having the ability to decompose fusaric acid and belonging to the genus Pseudomonas, microbial cells, crushed microbial cells thereof and/or extract of the microbial cells to fusaric acid. CONSTITUTION:A microorganism, having the ability to decompose fusaric acid which is a bacterium of the genus Pseudomonas, particularly preferably Pseudomonas cepacia UK-1 strain (FERM-P No. 9160), microbial cells thereof crushed microbial cells thereof and/or extract of the microbial cells are used to decompose fusaric acid produced by metabolism of fungi of the genus Fusarium. Even if soil or plant bodies are infected with fungi of genus Fusarium, the development of blight, such as fusarium wilt, etc., can be prevented or further progress of infectivity can be inhibited.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a microorganism capable of decomposing fusaric acid and a method of decomposing fusaric acid using the microorganism.

(Conventional technology) Diseases that cause growing plants to wilt and die 2 Examples include vine splitting disease of cucurbits such as cucumbers, watermelons, and melons, wilt of tomatoes, half blight of eggplants, yellowing of strawberries, and dryness of konjac. Rot disease, spring bald disease of grass, etc.

is known to be caused by infection with Fusarium N. The Fusarium genus bacteria that cause such infections include Fusarium oxysporum (Fusarius ■■赳■L) + Fusarium moniliforme (Pusarium soni).
There are many types of bacteria such as liform). These bacteria are soil contaminants and are absorbed from the soil into the plant body.
Migrate through conduits. What causes plants to die?

It is thought to be fusaric acid produced by the metabolism of these bacteria. Fusaric acid is produced by almost all plant pathogenic bacteria belonging to the genus Fusarium, including the two types of fungi of the genus Fusarium. This fusaric acid acts as a non-specific toxin and exhibits harmful effects not only on the host plant but also on other plant individuals. Fusaric acid increases the permeability of plant plasma membranes.

As a result, Ca'', K'', and N were deposited on the surface of the plant body such as nine leaves.
The interstitial fluid containing cations such as ``a'' and various amino acids exudes, and when this dries, the osmotic pressure on the surface of the plant increases, further increasing water transpiration.

As a result, the plants will wilt and die.

As a drug to prevent Fusarium infections.

For example, an 0-phenylenediamine derivative (trade name Nidotubudin-M) having the following structural formula is used as a soil fungicide.

(Left below) This chemical has the effect of killing Fusarium bacteria living in the soil, but it is not effective just by spraying it on the soil surface, so it must be mixed with the soil before sowing or planting plants. (There is a need.

Such work requires a great deal of effort. moreover.

The drawback is that they have little effect on preventing the progression of the disease after the symptoms of the infection appear.

On the other hand, there are tomato varieties that are resistant to infections caused by Fusarium fungi. In this tomato plant tissue,
As shown below, fusaric acid (1) is metabolized and decomposed, and N
-Methylfusaric acid amide (II). Furthermore, this fusaric acid decomposition effect is greater in cultivars with strong fusarium resistance.

CH2 (1) (n) If fusaric acid decomposes and disappears in this way.

It is thought that dehydration symptoms in plants due to water metabolism disorders can be prevented. For example, if microorganisms capable of decomposing fusaric acid are obtained, it is thought that the occurrence of infectious diseases caused by Fusarium bacteria can be avoided.

(Problem that the invention attempts to solve) The present invention solves the above-mentioned conventional drawbacks.

Another object of the present invention is to provide microorganisms and methods effective for preventing or inhibiting plant diseases such as vine rot and wilt caused by Fusarium fungi. The object of the present invention is to provide a microorganism capable of degrading fusaric acid produced by the metabolism of microorganisms of the genus and a method for decomposing fusaric acid using the microorganism.

(Means and effects for solving the problems) The microorganism of the present invention belongs to the genus Pseudomonas and has the ability to decompose fusaric acid, thereby achieving the above object. The fusaric acid decomposition method of the present invention can be carried out using, for example, the above-mentioned microorganisms.

The method includes contacting the bacterial cells, the crushed bacterial cells and/or the bacterial cell extracts with fusaric acid.

Thereby, the above objective is achieved.

The microorganism of the present invention is a bacterium of the genus Pseudomonas, and Pseudomonas cepacia is particularly desirable. Among these, particularly preferred strains are Pseudomonas cepacia U and 11 strains, which were isolated from soil by the inventor. In Japan, based on the mycological properties described below, the Verge Eighth Manual of Systematic Bacteriology Volume 1 (
Bergey's Manual of Systema
tic Bacteriology Volume 1
) Identified with reference to J.

Table 1 shows the mycological properties of the Pseudomonas cepacia UK-1 strain of the present invention.

From the above mycological properties, this strain belongs to Pseudomonas cepacia, Pseudomonas cepacia UK.
The strain was named as -1 strain (Huikoken Bacterial Serial No. 9160).

Dark cultivation medium does not need to be special, potassium, sodium,
Phosphates and sulfates such as magnesium.

Fusaric acid and various carbohydrates are added as carbon sources to a minimal medium containing chloride and the like. In order to enhance the ability of this microorganism to decompose fusaric acid, it is desirable that the culture medium contains fusaric acid as a carbon source or nitrogen source. Nitrogen sources used in this minimal medium include fusaric acid and nitrate.

Examples include inorganic nitrogen such as ammonium salts and organic nitrogen such as amino groups. It is also possible to add cornstarch liquor, yeast extract, etc. as a nutritional source, if necessary.

The culture temperature is 15-40°C, preferably 25-30°C. Culture pH is 4.0-9.0. Preferably 5.5-6
．． 5, and culture is performed aerobically with stirring or shaking for 1 to 5 days. Culture on a solid medium (agar medium) is also possible.

By growing the microorganism in the above-mentioned suitable medium and culturing it aerobically in a normal medium containing fusaric acid, the microorganism contained in the medium at a rate of up to 2000 ppm (μg 7 ml) Fusaric acid is almost completely degraded.

Fusaric acid decomposition by the microorganism of the present invention can also be carried out by growing the microorganism in advance in an appropriate medium.

It can be carried out using the resting bacterial cells, crushed bacterial cells, or bacterial cell extracts. The reaction system at that time is shown in Table 2.

Table 2 The amount of bacterial cells, crushed bacterial cells, or bacterial cell extracts used is about 1 to 10% by weight (net) of the entire reaction system, preferably around 5% by weight (net). Obtained by processing the body using an ultrasonic crusher or French press. The bacterial cell extract obtained by centrifuging the crushed bacterial cells may have a zero reaction pH in the range of 4 to 9. Preferably, it is adjusted to a range of 5 to 7.

The concentration of fusaric acid is below 20 QOppm, preferably in the range of 50 to 300 ppm. As the buffer solution, for example, a 50 mM phosphate buffer solution is used.

The reaction temperature may be within the range of 15 to 40°C, preferably 25 to 30°C. When using resting bacterial cells.

Fusaric acid can be almost completely decomposed without significant growth of bacterial cells. The reaction time varies depending on the bacterial cells, fusaric acid concentration, temperature, pH, etc., but usually takes from several minutes to 72 hours. be. The amount of fusaric acid in the system is calculated by measuring the absorbance (ODzt.), for example.

The primary strain of Pseudomonas cepacia l has the property of decomposing the above-mentioned fusaric acid, as well as the property of suppressing the growth of plant pathogenic bacteria. This strain and the plant pathogenic bacteria shown in Table 3 were cultured face-to-face in various media, and the degree of inhibition of the growth of the plant pathogenic bacteria was evaluated in 6 grades (-1±,
+, +, +1+, N). The results are shown in Table 3.
ZD), Czapek medium (Z), potato dextrose medium (PDA), potato shake medium (PS)
Using the flat plate medium of A), pathogenic bacteria were inoculated in the center of the medium, and UK-1 strain was inoculated at equal intervals in 4 areas around the 2nd periphery and cultured.

In the above evaluation.

- indicates a state in which plant pathogenic bacteria proliferate on the surface of the medium, and its growth is not inhibited at all, and 2 indicates a state in which growth is slightly inhibited. Tatami indicates a state in which plant pathogens are completely inhibited and cannot grow at all.

(Margin below) (Example) The invention will be explained below with reference to examples.

Soil from Kowakae, Higashi-Osaka City was suspended in sterilized water, the resulting supernatant was diluted, and applied to the screening medium shown in Table 4 (using fusaric acid as a mono-C source) at 30°C. The cells were cultured for 48 hours.

Table 4 The resulting colonies were subcultured onto the same medium, and the mycological properties of the resulting strain were examined and found to be the same as in Table 1, confirming that this strain was Pseudomonas cepacia UK-1 strain.

This Shademonas cepacia UK-1 strain was inoculated into the medium shown in Table 4 (liquid medium excluding agar).

Shaking culture was performed at 30°C for 3 days. The culture solution was centrifuged, the supernatant was collected, and the absorbance at 270 μm was measured. The absorbance of a medium (control medium) shaken under the same conditions without separate inoculation was measured after centrifugation, and the respective fusaric acid concentrations were calculated. The concentration of fusaric acid in the control medium was 100μg/-, whereas the concentration of fusaric acid in the culture solution of the two strains was 0.3μg/-.
It was god.

Implementation Group 1 Add 2 g/I of glucose to the medium shown in Table 4 (liquid medium excluding agar)! Pseudomonas cepacia UK-1 strain was grown in this medium.

The bacterial cells were collected by centrifugation. This was added at a ratio of 5% by weight (■at) to physiological saline containing fusaric acid at a ratio of 100μg/++d, and heated at 30°C for 24 hours.
Shake for an hour. The amount of residual fusaric acid in the solution is 7μg/+
It was d.

1m Pseudomonas cepacia 10'/11 plants/
- It was suspended in water (purified water) at a ratio of -. Water (purified water) was prepared as a control. Five cut tomato branches at the 5th to 6th node stage were used, and the cut ends were immersed in each of the above bacterial suspensions and water for 3 hours. After washing the cut with water, apply 300 μgo
It was immersed in an aqueous fusaric acid solution of about 20°C to absorb 10°. After washing the cut portion with water, the cut portion was immersed in water and left at 20°C.

When the above-mentioned water was absorbed, all the leaves showed wilting symptoms 8 to 16 hours after the start of immersion in the fusaric acid aqueous solution, and the stems and leaf veins turned brown and died. However, when the UK-1 strain suspension was absorbed, no abnormality was observed in any of the five bottles even after 72 hours.

Shademonas cepacia tJK-1 strain was cultured using the medium shown in Table 4 (liquid medium excluding agar). At 0.24.48 and 72 hours after the start of culture, a portion of the culture solution was taken and centrifuged at 3000G for 10 minutes, and the supernatant portion was collected. This was then filtered through a 0.2μ Millipore filter. The obtained filtrate and MS medium (Mura
Shige-Skoog medium: Physiol, P
lant.

15, 473 (1962)) at a volume ratio of 1:9.

Tomato (L co ersicon esc)
Free single cells of callus derived from the cambial region of the stem of ulenfum Millcv (Zuiko) were isolated at 10'.
The cells were added to a concentration of ~10' cells/- and left at 25°C for 24 hours. After 24 hours, callus cells were fluorescently stained with fluorescein diacetate (FDA). This was visually observed using a fluorescence microscope.

As a control for calculating the survival rate (%) of callus cells (n-500), MS medium was used instead of the above mixture. The results are shown in Table 5.

Table 5 (Effects of the Invention) According to the present invention, Pseudomonas bacteria having the ability to decompose fusaric acid are thus provided. When this microorganism, its bacterial cells, its bacterial cell fragments, and/or its bacterial cell extracts are applied to soil or plants, the production of Fusarium spp. even if the soil or plants are infected with Fusarium spp. Because fusaric acid is decomposed, diseases caused by infection with Fusarium species, such as vine wart and wilt, do not occur. Even when applied to plants or soil that have already developed an infection, fusaric acid is decomposed and inactivated, suppressing the further progression of the infection.

Furthermore, Pseudomonas cepacia UK-1 strain.

In addition to its ability to decompose fusaric acid, it has the ability to inhibit the growth of other plant pathogens, so it can be widely used to control plant diseases.

that's all

Claims (1)

[Claims] 1. A microorganism belonging to the genus Pseudomonas and having the ability to decompose fusaric acid. 2. Pseudomonas cepacia (￥Pseudomon
Claim 1 which is (as￥￥cepacia￥)
Microorganisms listed in section. 3. The microorganism according to claim 2, which is Pseudomonas cepacia UK-1 (Feikoken Bacteria No. 9160) strain. 4. A method for decomposing fusaric acid using a microorganism belonging to the genus Pseudomonas, its cells, its crushed cells, and/or its cell extract. 5. The microorganism is Pseudomonas cepacia (￥Pse
udomonas￥cepacia￥). 6. The microorganism is Pseudomonas cepacia UK-1 (
Claim No. 5, which is the strain
The method described in section.