JP2873931B2 - Mushroom disease control agent and control method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel strain having an ability to control mushroom diseases, a disease controlling agent and a method for controlling the disease using the same.

[0002]

[Prior Art] Pseudomonas
tolaasii ) produces the toxin trassin and causes great damage to various mushrooms such as oyster mushroom, enokitake, shiitake and mushroom. Pseudomonas tolaasii S8501 strain isolated from Japanese oyster mushroom
It produces eight toxins such as tracin I and II as virulence factors. These toxins not only cause lesions on mushrooms,
It is known that it has a specific activity of remarkably blackening potato slices (Shirata et al., 1995 Annual Report on Plant Disease 61:
493-502). In recent years, the image of mushrooms as a healthy food has increased, and most of the demand has been met by artificial cultivation. Artificial cultivation of mushrooms is performed by a semi-aseptic method so as not to be contaminated by such pathogenic bacteria. However, it is difficult to completely prevent the invasion of pathogens, and once the disease spreads, it leads to collapse of the production area, which is a serious problem. Further, since mushrooms eat fruit bodies, high safety is required for pesticides used for mushrooms. Furthermore, in view of the problem of environmental pollution caused by chemical pesticides in recent years, development of disease control agents that do not rely on chemical pesticides, that is, biological pesticides has been desired. However, there is no one that has been practically used as a highly effective control agent that guarantees safety against mushroom diseases.

[0003]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above situation, and has been developed in view of the above-mentioned situation.
An object of the present invention is to provide a means for safely controlling mushroom diseases caused by Pseudomonas tolaasii .

[0004]

The present inventors have SUMMARY for achieving] As a result of intensive studies to solve the above problems, a novel bacterial strain isolated from a fruiting body of Pleurotus ostreatus are Pseudomonas Torashii a pathogen mushroom (Pseudomonas Tolaasii ) inhibited the production of the toxin tolasin and found that it exhibited an excellent control effect on mushroom diseases, and thus completed the present invention.

[0005] That is, the present invention is a mushroom disease controlling agent comprising as an active ingredient a microorganism capable of controlling mushroom diseases caused by Pseudomonas tolaasii . The present invention is also a method for controlling mushroom disease, which comprises spraying the above-mentioned mushroom disease controlling agent on mushrooms.

Further, the present invention is a novel bacterium strain S9405 having an ability to control mushroom diseases. Hereinafter, the present invention will be described in detail.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The microorganism used in the present invention is a microorganism having an ability to control mushroom diseases caused by Pseudomonas tolaasii .
Although not particularly limited, a preferred strain is a novel bacterium S9405 strain isolated from the fruiting bodies of cultivated Pleurotus ostreatus. This strain was deposited with the National Institute of Advanced Industrial Science and Technology under the deposit number FERM.
P-15472 (Original deposit date: February 23, 1996).

In order to isolate the S9405 strain, first, a commercially available oyster mushroom is inoculated with P. tolaasii, a rot fungus of the oyster mushroom.
It becomes evident (induced / proliferated) by causing disease. Confirmation of the manifestation is performed by placing the diseased oyster mushroom on a potato section and checking whether the potato turns black two days later. P. t
If the black discoloration of the potato caused by olaasii is completely inhibited, it is judged that the target bacterium has grown, and a single colony is separated from the oyster mushroom by a conventional method. Then the isolated strain
Potato sections are inoculated with P. tolaasii , and only strains that completely inhibited black discoloration are selected.

The strain isolated as described above is P. t
It almost completely inhibits the production of the toxin tolasin, the pathogen of olaasii , and this inhibitory action is highly specific. In addition, the strain has the property of not inhibiting the growth of P. tolaasii .

[0010] For culturing the S9405 strain, it is not necessary to use a special method, and a method similar to known aerobic bacteria can be used. As the medium, any synthetic medium or natural medium can be used as long as the medium appropriately contains assimilable carbon sources, nitrogen sources, inorganic substances, and necessary growth promoting substances. Specific examples of the medium include a potato semi-synthetic medium, an oyster mushroom medium, a King B medium, a peptone medium,
Potato sucrose medium and the like can be mentioned. During culturing, the temperature is 15 to 33 ° C, preferably 25 to 28 ° C.
C., pH 6.0-8.0, preferably 7.0-7.5.
It is desirable to maintain Under the above conditions,
After culturing for about 2 days, a sufficient amount of colonies are formed on the surface of the medium.

When the present strain is used in a control method, microbial cells or metabolites thereof (eg, cell membrane proteins, peptidoglycan) are used, but microbial cells are preferably used. The cells can be recovered by pouring sterile water onto the colonies and scraping the surface of the medium with a brush. In the case of liquid culture, it can be used as it is or collected by a centrifuge.

[0012] The control method is carried out by spraying the microbial cells or metabolites thereof directly on mushrooms or on a fungal bed, a cultivation facility, a harvesting container, a packaging pack, or the like. The disease control agent of the present invention may be used directly as a microbial cell, or a metabolite thereof, but is generally mixed with a solid or liquid carrier usable for agrochemicals,
It is prepared and used in the form of wettable powders, powders, granules, emulsions, oils, capsules and the like.

[0013] The concentration of the microbial cells is 1 x
10 ⁵ to 1 × 10 ⁹ / ml, preferably 1 × 10 ⁷ to 1 × 10
^It is appropriate to use ⁹ cells / ml. In the present invention, the mushrooms at the time of treatment are preferably in the fruiting body forming stage to the harvesting stage. Diseases to be controlled according to the present invention include oyster mushroom rot,
Enokitake black rot, mushroom browning, shiitake black rot and the like can be mentioned.

[0014]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to examples and test examples, but the present invention is not limited to these examples. [Example 1] Separation of bacteria Separation source: Commercial oyster mushroom was used. Separation medium: Potato semi-synthetic medium (potato tuber
300g of decoctions _{1 L, Ca (NO 3)} 2 · 4H 2 O 0.5g, Na 2 HPO 4 · 1
(2H ₂ O 2.0 g, peptone 5 g, sucrose 15 g, agar 15 g)
And King B medium (peptone _{20g, K 2 HPO 4 1.5g,} MgS
O ₄ · 7H ₂ O 1.5g, glycerin 10 ml, agar 15 g, distilled water 1
L) was poured into a petri dish and used as a flat plate.

[0015] Induction of bacteria: P. tolaasi added to commercial oyster mushrooms
i S8501 strain (Oyster mushroom rot fungus isolated from Oyster mushroom,
Inoculated at a concentration of 10 ^8-9 cells / ml and stored in a moist chamber at 20-25 ° C for 4-8 days, infecting the disease with the disease. Bacteria also grew and became separable.

Confirmation of bacteria: The diseased oyster mushroom is placed on a potato section and kept for 1 to 2 days. P. tolaasii
Produces toxins and turns potatoes black, but oyster mushrooms grown with toxin-inhibiting bacteria no longer discolor potato slices. In such oyster mushrooms, toxin production inhibiting bacteria induce and
It was determined that they had proliferated.

Isolation of bacteria: Various types of bacteria are isolated as a single colony from oyster mushrooms, for which induction of bacteria has been confirmed, by a conventional method. Each isolated strain and the P. tolaasii S8501 strain were mixed and inoculated into potato slices, and strains that completely inhibited the potato blackening caused by the P. tolaasii S8501 strain were selected and determined as the target toxin production-inhibiting bacteria.

Preservation of bacteria: Among the bacteria isolated by the above method, S9405 strain was used as a standard strain having an inhibitory effect on toxin production, and was deposited with the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology under the deposit number FERM P.
-15472 (Original deposit date: February 23, 1996).

[Test Example 1] Action characteristics of the isolated bacterial S9405 strain The bacterial S9405 isolated in Example 1 was subjected to P. tol
The effects on aasii growth and toxin productivity and activity were selected. As a result, the following became clear.

The isolated bacterium strain S9405 does not inhibit the growth of P. tolaasii . When the isolated bacterium S9405 was mixed with P. tolaasii and inoculated into potato slices, the potato blackening effect of P. tolaasii was completely inhibited. The effect was extremely strong, and even when the isolated bacteria were cultured at a 1/1000 ratio in a liquid medium, the production of tracin was completely inhibited.

Mixing P. tolaasii with the culture filtrate of the isolated bacteria or a 20-fold concentrated solution thereof and inoculating it into potato sections did not inhibit black discoloration. This indicates that the culture filtrate of the isolated bacteria does not contain the blackening inhibitor. Mixing and inoculating P. tolaasii culture filtrate (containing the toxin tolasin) with the isolated bacteria did not inhibit potato section blackening. This indicates that the isolated bacteria do not have the function of detoxifying or inactivating tracin.

[Test Example 2] Pest control effect of Pleurotus rot fungus Spraying isolated bacteria S9405 strain and pathogen P. t to be inoculated
cell concentration of olaasii S8501 strain were adjusted to both 10 ⁸ / ml. Before and after spraying S9405 strain on Oyster mushroom 0,3,6,2
P. tolaasii S8501 strain was inoculated 4 hours and oyster mushrooms 4
Keep in wet room for ~ 5 days. As a control, an inoculated plot of only P. tolaasii S8501 strain was provided without spraying S9405 strain. As a result, P. t before control and S9405 strain application 6, 24 hours before application
The disease occurred in oyster mushrooms in the olaasii- inoculated plots , but did not occur in the other plots . From this result, S9
Spraying 405 strains did not have any therapeutic effect, but it was determined to have a preventive effect and a disease-suppressing effect.

[0023]

[Table 1] Suppression effect ^{(* 1) (* 2) of} oyster mushroom rot caused by isolated bacteria S9405 and relationship between spraying time

^{* 1) The} disease was observed 4 to 5 days after the time of spraying the isolated bacteria. ^{* 2)} Degree of onset Four stages of severe (++), medium (+), weak (±), and no (-) ^{* 3) The-} mark attached to the time means before the time of inoculation of the pathogenic bacteria.

The following are formulation examples. 10 cells
It was used at a dilution of 0 to 1000 times. Formulation Example 1 (Solution) S9405 strain cells (1 × 10 ¹⁰ ) were added per 1 ml of sterilized water and mixed to prepare a solution.

Formulation Example 2 (Wettable powder) 9% maltose, 1% clay, 90% water S9 / ml
The 405 strain cells (1 × 10 ¹⁰ cells) were suspended. After air-drying, the dried product was mixed and pulverized to prepare a wettable powder.

Formulation Example 3 (Powder) S9 / g of a mixture of hydroxypropyl-β-cyclodextrin 14%, white carbon 12% and clay 74%
The 405 strain cells (1 × 10 ¹⁰ cells) were added and mixed. After drying, the powder was uniformly mixed to obtain a powder.

Formulation Example 4 (Granules) S9405 strain cells (1 × 10 ¹⁰ cells) per 1 g of a mixture of 15% β-cyclodextrin, 2% starch, 18% bentonite, 36% calcium carbonate, and 29% water Was added and kneaded, and the mixture was granulated with a granulator and dried to prepare granules.

Formulation Example 5 (Emulsion) Polyoxyethylene nonyl phenyl ether ammonium phosphate 18%, polyoxyethylene nonyl phenyl ether 6%, triethyl phosphate 29%, tolubutyl phosphate 47%
The S9405 strain cells (1 × 10 ¹⁰ cells) were added per 1 g of the mixture to uniformly suspend to prepare an emulsion.

[Formulation Example 6] (Oil) Spindle oil 95%, Castor oil 4%, Silicone oil
S9405 strain cells (1 × 10 ¹⁰ cells) were added to 1 ml of a 1% mixed solution and uniformly suspended to prepare an oil solution.

Formulation Example 7 (Capsule) 0.7% sodium alginate, 5% kaolin, 15 glycerin
%, 79.3% water in 1 ml of a mixed solution (1 × 10 ¹⁰
Were added and suspended, and added dropwise to 0.2 M calcium acetate to obtain a capsule-like product. This was air-dried to prepare a capsule.

[0032]

Industrial Applicability The present invention provides a novel disease control agent and method for mushrooms. The disease control agent and the control method of the present invention utilize microorganisms that are isolated from cultivated oyster mushrooms and are considered to be attached to or endogenous to the fruiting body, so there is no concern about environmental pollution, and edible mushrooms It does not adversely affect the safety of the vehicle. Therefore, it is extremely useful industrially.

Claims (4)

(57) [Claims] [1] Pseudomonas
A fungicide for mushrooms, which comprises, as an active ingredient, a microorganism capable of controlling mushroom diseases caused by tolaasii ). 2. Pseudomonas
2. The agent for controlling mushroom diseases according to claim 1, wherein the microorganism capable of controlling mushroom diseases caused by M. tolaasii ) is a novel bacterium S9405. 3. The mushroom disease controlling agent according to claim 1 or 2,
A method for controlling disease of mushrooms, which is sprayed on mushrooms. 4. A new bacterium strain S9405 having a controlling ability against mushroom diseases.