JP2002176969A - New yeast, and plant disease injury controlling agent and method for controlling plant disease injury - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a biological agrochemical using a new kind of yeast that does not give influence to plants and is capable of inhibiting both the outbreak and propagation of plant disease injuries. SOLUTION: The new kind of yeast exhibits a controlling action on plant disease injuries unlike that of yeast having the known base sequence in 26S rDNA D1/D2 domain. The plant disease injury controlling agent uses the above yeast and the method for controlling plant disease injuries uses the above controlling agent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel yeast, a plant disease controlling agent and a plant disease controlling method using the same.

[0002]

2. Description of the Related Art For stable production of agricultural products, the use of pesticides is indispensable on agricultural land. However, various adverse effects have appeared since a large amount of chemically synthesized pesticides containing chemical substances that do not exist in nature have been sprayed on agricultural land for a long period of time. The most important issue is the impact on humans and environmental organisms. In recent years, the toxicity of non-target organisms has been reduced due to the dramatic improvement in selective toxicity of synthetic pesticides to organisms, but the potential danger of synthetic pesticides continues to exist. In particular, there is a concern that highly toxic by-products mixed into chemically synthesized pesticides may affect non-target organisms. In fact, a serious problem was that trace amounts of dioxin, a toxic substance mixed as a by-product in herbicides, were accumulated in river sludge and bio-enriched in fish and shellfish.

[0003] In order to solve these problems, it has been desired to protect the crops in an environmentally-friendly manner by reducing the use of synthetic chemical pesticides.
Reduce the use of chemically synthesized pesticides by performing comprehensive control that rationally combines chemical control, biological control, ecological control, cultivated control and preventive control. Attention has been paid to the reduction. Biological control using microbial materials is an effective means.

[0004] As a commercially available microbial fungicide, in Japan, a botokiller (Idemitsu Kosan Co., Ltd.) containing Bacillus bacteria for controlling gray mold and a biokeeper containing non-pathogenic bacteria of Chinese cabbage soft rot for controlling Chinese cabbage soft rot. (Central Glass Co., Ltd.). In foreign countries, AQ-10 (Ecogen, USA) containing Ampelomyces quisqualis to control grape powdery mildew, Trichoderma harzianum and T. poly to control storage diseases of flowers, fruits and vegetables
Binab T containing sporum (Bio-Innovat, Sweden)
ion), Trichoderma harzi to control vegetable soil disease
Trichodex containing anum (Makhteshim, Israel)
Corporation) is on the market.

As a bactericide containing yeast as an active ingredient, Ca
Aspire (Ecogen) for controlling storage diseases of citrus fruits and apples using ndida oleophila is on the market. Also,
As a research report, there is a method of controlling gray mold of cucumber and tomato using Aureobasidium pullulans (AJ Dik
et al., European Journal of Plant Pathology 105: p
p. 115-122 (1999))

[0006]

To obtain a sufficient effect against gray mold, Trichoderma harzianum (Tri
chodex) and Aureobasidium pullulans have a high application spore (cell) density of 10 ⁷ spores (cells) / ml. For powdery mildew, Ampelomyces quisqualis (AQ-10)
Although the applied spore density is as low as 10 ⁶ spores (cells) / ml, A. quisqualis forms a black spore mass on leaves after being infected with powdery mildew, and has the disadvantage that the leaves look dirty. An object of the present invention is to provide a biological pesticide using a novel yeast that does not adversely affect plants and suppresses the occurrence and spread of plant diseases.

[0007]

Means for Solving the Problems The present inventor has collected yeasts inhabiting the leaves of plants from all over Japan, and isolated from them yeasts exhibiting a controlling effect on plant diseases. Based on the above, the present invention has been completed.

[0008] That is, the present invention relates to a yeast exhibiting a controlling effect on plant diseases, wherein the base sequence of the D1 / D2 region of 26S rDNA has any one of the following bases (1) to (4): The sequence is related to yeast. (1) A nucleotide sequence showing 99% or more homology with the nucleotide sequence described in SEQ ID NO: 1 (2) A nucleotide sequence showing 94% or more homology with the nucleotide sequence described in SEQ ID NO: 2 (3) A nucleotide sequence shown in SEQ ID NO: 3 A nucleotide sequence showing 98% or more homology with the nucleotide sequence (4) A nucleotide sequence showing 98% or more homology with the nucleotide sequence described in SEQ ID NO: 4

[0009] The present invention also relates to a plant disease controlling agent comprising the above yeasts as an active ingredient. Furthermore, the present invention relates to a method for controlling plant diseases using the above yeasts.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The yeasts of the present invention show a controlling effect on plant diseases,
The nucleotide sequence of the D1 / D2 region of the 26S rDNA has the following (1)
Or (4).

(1) a nucleotide sequence showing 99% or more homology with the nucleotide sequence described in SEQ ID NO: 1 (2) a nucleotide sequence showing 94% or more homology with the nucleotide sequence described in SEQ ID NO: 2 (3) SEQ ID NO: A nucleotide sequence showing 98% or more homology with the nucleotide sequence described in No. 3 (4) A nucleotide sequence showing 98% or more homology with the nucleotide sequence described in SEQ ID NO: 4

Generally, yeasts represent fungi that grow on a single cell. However, yeast is not a formal taxonomy, but a group of taxonomically unrelated fungi that exhibit a growth morphology that grows on a single cell. Fungi that grow on a single cell during part of the life cycle or under certain circumstances are sometimes referred to as yeast. Therefore, also in this specification, the term "yeasts" refers to fungi having a process of growing and growing in a single cell.

The nucleotide sequence of the D1 / D2 region of the 26S rDNA of the yeast of the present invention may be any one as long as it satisfies the above conditions. Preferred nucleotide sequences are described in SEQ ID NOs: 1 to 4. Can be exemplified. As yeasts having such a base sequence, JT-1165 strain, JT-
1209 strain, JT-1211 strain and JT-1213 strain can be mentioned.
The JT-1165, JT-1209, JT-1211, and JT-1213 strains have been deposited with the Ministry of International Trade and Industry at the National Institute of Advanced Industrial Science and Technology under the accession numbers FERM BP-7175, 7176, 7177, and 7178. (Deposit date: June 1, 2000).

JT-1165, JT-1209, JT-1211, JT-12
The morphology and properties of the 13 strains are as shown in Examples 4 and 5 below. These morphologies and properties, as well as the 26S rDNA
From the nucleotide sequence of the D1 / D2 region, JT-1165 strain, JT-1211 strain, and JT-1213 strain belong to the genus Aureobasidium . The JT-1209 strain has not been identified.

The yeasts of the present invention can be obtained by selecting yeasts inhabiting the leaves of plants from the yeasts inhabiting the leaves of the plants, using the control action against plant diseases as an index, as shown in the examples below.
Also, since the D1 / D2 region of 26S rDNA can be used for yeast identification and classification (CPKurtzman et al., Antonie van L.
eeuwenhoek 73: pp.331-371, 1998), SEQ ID NOs: 1-4.
The yeasts of the present invention can also be obtained by selecting using the nucleotide sequence of the D1 / D2 region of the 26S rDNA described in (1) as an index.

For culturing the yeasts of the present invention, it is not necessary to use a special method, and a usual method for culturing yeast can be used. The medium includes assimilable carbon sources, nitrogen sources,
Any of a synthetic medium and a natural medium can be used as long as the medium appropriately contains an inorganic substance and a necessary growth promoting substance. Specific examples of the medium include YM agar medium (Difco
Co., Ltd.), potato dextrose agar medium (Difco) and the like. When culturing, keep the temperature at 15-30.
C., preferably at 25 to 30.degree. By culturing for about 2 to 3 days under the above conditions, a sufficient amount of cells can be obtained. The method of mass culture is
This is as shown in Example 7.

The yeast of the present invention may be used as a plant disease controlling agent or a plant.
It can be used for disease control methods. Control agent of the present invention
May be used directly as yeast cells,
Mix with a solid or liquid carrier that can be
Formulation forms such as Japanese preparations, powders, granules, emulsions, oils, capsules, etc.
It can also be used after being prepared. Yeasts in control agents
Concentration of 10⁸Cells / ml ~ 5 × 10⁹Cells / ml is preferred
5 × 10⁸Cells / ml ~ 10 ⁹More preferably, cells / ml
New

The plant disease to be controlled is not particularly limited, and for example, powdery mildew of cucurbits (scientific name: Sphaer
otheca fuliginea ), gray mold of various vegetables and flowers (pathogenic fungi name: Botrytis cinerea ), tomato powdery mildew (pathogenic fungi name: Oidium violae, etc.), strawberry powdery mildew (pathogenic fungal name: Sphaerotheca macularis ), rose powdery mildew ( Pathogen name: Sphaerotheca ponnosa ), but is not limited thereto.

The yeasts of the present invention can exert a controlling effect by a use mode such as spray application to a plant. The amount of yeast used depends on the mode of use, but when it is applied to a field, it is preferable to apply it so that the number of yeast cells per field 10a is 10 ¹¹ to 2 × 10 ¹³ .

[0020]

EXAMPLES [Example 1] Isolation of plant leaf surface yeast Healthy tomato and cucumber plant leaves were collected from all over Japan. After cutting these plant leaves into sections of about 1 cm × 1 cm, 40 sections were shaken in 10 ml of sterile 0.02% Tween 20 aqueous solution for about 3 hours. 50 μl of the supernatant was spread on a potato dextrose agar medium supplemented with chloramphenicol (75 μg / ml) and streptomycin sulfate (125 μg / ml) and cultured at 25 ° C. The culture medium was observed from day 4 to day 8 of culture, and yeasts were isolated.

[Example 2] Control effect of yeast on cucumber powdery mildew and cucumber gray mold A conidial spore suspension (2 × 10 ⁴ spores / ml) of powdery mildew fungus was applied to the first leaf of cucumber. 2 ml was sprayed and inoculated per pot. Three
One day later, a suspension of the yeasts isolated in Example 1 (10 ⁸ cells / m
l) was inoculated by spraying 2 ml per pot, and then placed in an inoculation box for 18 hours. Thereafter, the pot was transferred to a greenhouse, and the number of powdery mildew colonies on the first leaf of the true leaf was counted 7 days after inoculation with yeast. Saccharomyces cervisiae IFO0565 strain was used as a control yeast.

Also, a conidia spore suspension of the fungus of Botrytis cinerea (5.
(× 10 ⁶ spores / ml) and 50 μl of the yeast suspension (10 ⁶ cells / ml) isolated in Example 1 were dropped on cucumber cotyledons.
Mixed. After placing the inoculated plants in a moist chamber, the lesion length on the leaves was measured 8 to 10 days later. Saccha as control yeast
romyces cervisiae IFO0565 strain was used. Among the yeasts isolated in Example 1, JT-1165 and JT-1165, which showed a high control effect against cucumber powdery mildew and cucumber gray mold
T-1209, JT-1211 and JT-1213 strains were selected. Table 1 shows the inhibition rates of powdery mildew and gray mold of each strain.

[0023]

[Table 1]

Example 3 Influence of yeasts on crops Cucumber, tomato, pepper, soybean, perilla, spinach, rice, wheat, barley and corn
After JT-1165, JT-1209, JT-1211 or JT-1213 cell suspension (10 ⁸ cells / ml) per pot 2ml sprayed and inoculated strains was 24 hours plated on inoculation box. Thereafter, the pot was transferred to a greenhouse, and the inoculated plants were observed until 21 days after the yeast inoculation. A water treatment section was provided as a control. As a result, there was no effect of the strains JT-1165, JT-1209, JT-1211 and JT-1213 on all crops.

Example 4 Morphology of Yeasts The morphology of strains JT-1165, JT-1209, JT-1211 and JT-1213 was examined visually or by microscope. The morphology of each strain is as follows. 1. On JT-1165 potato dextrose agar, the initial colonies were white paste. Thereafter, the colonies became creamy and hyphal growth was observed from around the colonies. The cells were elliptical and had a size of about 3-10 × 2-5 μm.

2. On the JT-1209 potato dextrose agar medium, the initial colonies were white paste. Thereafter, vigorous mycelial growth was observed. In long-term culture, colonies sometimes became black. The cells were elliptical and had a size of about 3-9 × 1.5-5 μm.

3. On JT-1211 potato dextrose agar, the initial colonies were white paste. Thereafter, the colonies became creamy and hyphal growth was observed from around the colonies. The colonies became black in long-term culture. The cells are oval,
The size was about 4-12 × 2-6 μm.

4. On JT-1213 potato dextrose agar medium, the initial colonies were white paste-like. Thereafter, the colonies became creamy and hyphal growth was observed from around the colonies. The colonies became black in long-term culture. The cells are oval,
The size was about 3-12 × 2-6 μm.

Example 5 Properties of Yeasts (Utilization, etc.) The properties of the isolated yeasts were examined with reference to the following literature.・ The yeasts a toxonomic study, third revised an
d enlarged edtion (NJW Kreger-van Rij, Elsevier
(Science Publishers BV, 1984) ・ The yeasts a toxonomic study, fourth revised a
nd enlarged edtion (CP Kurtzmann and JW Fell,
Elsevier Science Publishers BV, 1998) YEASTS: Characteristics and identification, se
cond edition (JA Barnett, RW Payne and D. Yarr
(ow, Cambridge University Press, 1990) ・ Classification and identification of revised microorganisms (edited by Takeharu Hasegawa, Gakkai Shuppan Center, 1984)

A fresh colony obtained by repeating inoculation and culturing twice or more on a YM agar medium was used as an inoculum. Also, the degree of bacterial growth in liquid culture was determined by Wickerham's scal
(3+: 3 / 4mm wide line is not visible at all. 2+: Lines appear to be scattered. 1+: Lines can be recognized.
The tip appears unclear. ).

(1) Fermentability Add 10-15 ml of 0.5% yeast extract (Difco
Was placed in a test tube, and a 50 mm × 6 mm Durham tube was submerged in the test tube, followed by autoclaving. 2. A 500 mM or 100 mM saccharide solution sterilized by filtration was added to a final concentration of 50 mM. 3. 10 ⁷ cells / ml ・ 0.5% yeast extract cell suspension
After inoculating 100 μl, the cells were cultured at 25 ° C. by standing. The test tube was stirred every day so that the precipitated cells became uniform in the culture solution. 4. The presence or absence of air in the Durham tube was observed until 28 days after the culture. If air accumulated by 7 days of cultivation, "+" was given. culture
28 days later, if air accumulates, it is rated "D".

(2) Carbon compound assimilation 1. A 10-fold concentration medium (3.35 g Bacto Yeast Nitrogen Ba
After preparing se (Difco), 2.5 g glucose / 50 ml)
Filter sterilized. For carbon sources other than glucose, 2.5 g glucose equivalent was added. 2. 0.5 ml and 4.5 ml of sterile water were placed in a test tube and mixed. 3. After inoculating 100 μl of a cell suspension of 10 ⁷ cells / ml in water, the cells were cultured at 25 ° C. with shaking. 4. After 7 days of culture, “+” was given on Wickerham's scale if it was 2+ or 3+, and “D” if it was 2+ or 3+ after 14 and 21 days of culture.

(3) Assimilation of nitrogen compounds Carbon Base Agar (11.7 g Bacto Yeast Carbon B
ase (Difco), 20 g washed purified agar (Difco
/ L) was prepared and dissolved in hot water. Each 15 ml was placed in a test tube, autoclaved, and then immersed in 45 ° C water. 2. One test tube was inoculated with 500 μl of a cell suspension of 10 ⁷ cells / ml water, which was allowed to stand at 25 ° C. for one week, and mixed well. After pouring 3.2 into a petri dish, it was left still for 30 minutes or more to obtain a plate medium (plate). 5 μl of a 4.2% nitrogen source solution (filter-sterilized) was dropped on the plate, and cultured at 25 ° C. (NH ₄ ) ₂ SO ₄ was used as a control. 5. Four days after the cultivation, if there was a zone of a clear dark bacterial colony, it was marked as "+", and if there was a zone that was slightly recognizable, it was marked as "W".

(4) Vitamin requirement 1. A 10-fold concentration of a vitamin-free medium and stock solutions of various vitamins were prepared and sterilized by filtration. 2.5 ml of a vitamin-free medium, a prescribed amount of a vitamin solution and sterile distilled water were added to make up to 50 ml. Each 5 ml was placed in an autoclaved test tube. 3. 50 μl of a cell suspension of 2.5 × 10 ⁶ cells / ml in water which had been allowed to stand at 25 ° C. for 1 week was inoculated and cultured at 25 ° C. with shaking. 4. After 3 days of cultivation, “+” was given on Wickerham's scale if 2+ or 3+, and “D” was given on 7+ days after culturing.

(5) Productivity of extracellular starch-like substance (NH ₄ ) ₂ SO ₄ 0.5 g, KH ₂ PO ₄ 0.5 g, MgSO ₄・ 7H ₂ O 0.2
5 g and glucose 5 g were dissolved in 250 ml of distilled water, adjusted to pH 4.5, and sterilized by filtration. 2. 1 was mixed with the autoclaved 4% agar (250 ml). 3. One drop of a 20% yeast extract solution and one drop of a 100-fold concentration of a vitamin solution were dropped into 2, mixed, and then a plate was prepared. 4. The bacteria were streaked and cultured at 25 ° C. for 1 and 2 weeks. 5.5 ml of Idione solution (166 mg I, 333 mg KI / 50
ml distilled water) was poured into the plate. "+" If blue
And

(6) Raw Acid 1.5 g Yeast Extract, 50 g dextrose, 5 g calcium carbonate, 20 g Bacto Agar (Difco) were dissolved in 1 L of distilled water and dissolved in 1 L of distilled water. And autoclaved. 2. After boiling in hot water of 50 ° C., the test tube was shaken well to disperse calcium carbonate to obtain a slope medium. 3. The bacteria were streaked and cultured at 25 ° C. 4. After 7 days of culture, if the culture medium becomes transparent, mark it as `` + '' and culture 21
A day later, when the medium became transparent, it was designated "D".

(7) Urea Hydrolysis Property 1.5.8 g Bacto Urea Agar Base (Difo) was dissolved in 20 ml of distilled water and sterilized by filtration. 2. 90 ml of distilled water was added to 1.5 g of Bacto Agar, dissolved in hot water, and placed in test tubes in 4.5 ml portions, followed by autoclaving. 3. 0.5 ml of 1 was placed in the test tube of 2, and stirred well to prepare a slant medium. 4. The bacteria were streaked and cultured at 25 ° C. 5. Observation was carried out every day until 5 days of culture.

(8) Growth in hyperosmolar medium (medium containing glucose) 1. 50% (w / w) glucose medium: 50 g of glucose was added to 50 g of glucose medium.
Was added to a 0.5% (w / v) Yeast extract solution and dissolved in hot water. 2.3% (w / v) Bacto Agar was dissolved in hot water. 1 '. 60% (w / w) glucose medium: 60 g glucose to 40 g
Was added to a 0.5% Yeast extract solution and dissolved in hot water. 2 '. 3% (w / v) Bacto Agar was dissolved in hot water. 3.2 5 ml of each of 2 and 2 ′ was placed in each of 1 and 1 ′ test tubes, and then autoclaved to prepare a slant medium. 4. The bacteria were streaked and cultured at 25 ° C. The mouth of the test tube was sealed with parafilm to prevent the medium from drying. 5. After 7 days and 21 days of culture, the presence or absence of bacterial growth was observed. When the bacteria grew by 7 days after the cultivation, the score was "+", and when the bacteria grew by 21 days, the score was "D".

(9) Growth temperature 1. The bacteria were streaked on a pre-warmed YM agar medium in an incubator at 25 ° C., 30 ° C., 35 ° C., 37 ° C., or 40 ° C., and then cultured at each temperature. . 2. After 3 days of culture, the presence or absence of bacterial growth was observed. If evaluation was not possible due to ambiguous colonies, the colonies were streaked on fresh YM agar and subcultured.

(10) Resistance to cycloheximide 1.6.7 g of Yeast Nitrogen Base (Difo) and 10 g of glucose were dissolved in about 80 ml of distilled water. 2. 0.1 g and 1 g cycloheximide / 2.5 ml acetone solutions were prepared. 3. Add 1 to 2, and add distilled water to make a total volume of 100 ml.
Filter sterilized. This was used as a 10-fold concentration basic medium. 4. 0.5 ml of 3 was placed in a test tube containing 4.5 ml of autoclaved distilled water and mixed. 5. After inoculating 100 μl of a cell suspension of 10 ⁷ cells / ml in water, the cells were cultured at 25 ° C. with shaking. 6. After 7 days of cultivation, “+” was given for Wickerham's scale if 2+ or 3+, and “D” for 2+ or 3+ after 21 days of cultivation.

(11) DBB (Diazonium Blue B) Color Reaction The bacteria were streaked on a YM agar medium and cultured at 25 ° C for 2 weeks. 2. The plate was heated to 55 ° C. for 16 hours and then cooled at room temperature. 3. DBB reaction solution cooled on ice (1 mg / ml 0.1 M TrisHCl
buffer (pH 7.0)) 4 ml was poured on the plate. 4. If the colony turned red within 2 minutes, it was marked "+".

The above results are shown in Tables 2 to 7. The properties of all strains did not completely match those of known strains.
However, the properties of JT-1165, JT1211, JT-1213 are Aure
It was similar to that of obasidium pullulans . Tables 2-7
The yeasts a toxonomic study, fourth revised and
enlarged edtion (CP Kurtzmann and JW Fell, El
A. pu according to sevier Science Publishers BV, 1998)
The properties of llulans are also described.

[0043]

[Table 2]

[0044]

[Table 3]

[0045]

[Table 4]

[0046]

[Table 5]

[0047]

[Table 6]

[0048]

[Table 7]

<Symbols in Tables 2 to 7> +: positive response-: negative response D: delayed positive response W: weak positive response NT: not tested

<Column symbols of A. pullulans in Tables 2 to 7> +: positive response-: negative response w: weak response x: positive or weak response v: variable (+/-, w /-) response

Example 6 Determination of Nucleotide Sequence of Yeast rDNA-D1 / D2 Region and Homology Search The single colony streaked on YM agar medium was mixed with 3 ml of YM
After inoculating broth (Difco) and culturing with shaking at 25 ° C for 24 hours, genomic DNA was extracted and purified from the cells using Gen Toru-kun (for yeast and Gram-positive bacteria) manufactured by Takara Shuzo Co., Ltd. . 50 μg genomic DNA, 10 pmol primer NL-1
(5'-GCATATCAATAAGCGGAGGAAAAG), 10 pmol primer
NL-4 (5′-GGTCCGTGTTTCAAGACGG) and sterile distilled water were mixed to make 25 μl. This is Amersham Pharmacia Biotech
In addition to Ready-To-Go PCR Beads manufactured by the company, a PCR reaction solution was prepared. In the PCR, after a reaction at 95 ° C for 5 minutes, a reaction at 95 ° C for 1 minute, 52 ° C for 2 minutes and 72 ° C for 2 minutes was performed in 36 cycles. Primer sequence, rDNA-D1 / D2 region amplification and purification method is Clet
us P. Kurtzman and Christie J. Robnett. Identifica
tion and phylogeny of ascomycetous yeasts from ana
lysis of nuclear large subunit (26S) ribosomal DNA
partial sequences. (Antonie van Leeuwenhoek 73: p
p.331-371, 1998).

After confirming the amplified DNA fragment by agarose gel (1.5%) electrophoresis, the amplified DNA fragment was purified using Centricon-100 manufactured by Amicon. Next, ABI's B
The sequencing reaction was performed using igDye Terminator Cycle Sequenceing Kit. NL-1, NL-2 as primer
A (5'-CTTGTTCGCTATCGGTCTC), NL-3A (5'-GAGACCGATAGC
GAACAAG) and NL-4. Ce from Applied Biosystems
After purifying the DNA solution with an ntri Sep spin column, the ABI Mod
Using el 310, both strands of the DNA fragment amplified by PCR
The DNA sequence was decoded. JT-1165, JT-1209, JT-1211 and JT
The nucleotide sequences of the rDNA-D1 / D2 region of the -1213 strain are shown in SEQ ID NOs: 1, 2, 3, and 4, respectively.

DNA homology search (National Center for
As a result of Biotechnology Information), there was no strain that matched the known bacteria. However, JT-1165, JT-1211 and JT
The nucleotide sequence of the -1213 strain is Aureobasidium pullulans ATC
There was high homology with C62921 strain. The DN of the rDNA-D1 / D2 region between JT-1165, JT-1211 and JT-1213 and the ATCC 62921 strain
The homology of the A sequence was 98.9%, 97.4% and 97.5%, respectively. Similarly, the nucleotide sequence of JT-1209 strain is Nectria vil
The highest homology with that of ior ATCC16217 was 93.8%. The above results are shown in JT-1165, JT-1209, JT-1211 and JT
The T-1213 strain is a new species or strain (strai
n).

[Example 7] Liquid culture of yeast Cells were mass-cultured by liquid culture. A JT-1165 colony cultured on a YM agar medium was inoculated into 100 ml of a YM broth medium in a 500 ml Erlenmeyer flask, and cultured with shaking at 150 rpm at 25 ° C. for 18 hours. In addition, 10 ml of this culture
(About 9 × 10 ⁷ cells / ml) in a 3 L Erlenmeyer flask
After inoculating into YM broth medium, the cells were cultured at 25 ° C. for 18 hours with shaking at 150 rpm. The number of cells in the culture solution was counted over time using a hemocytometer. As a result, after 24 hours of culture, cell growth reached a stationary phase. The maximum cell density in the culture was about 9 × 10 ⁷ cells / ml.

Example 8 Preparation of Yeast Preparation Example 1 (Wettable Powder) 10 ⁹ cells (JT-1165) were suspended per 1 ml of a mixed solution of 9% maltose, 1% clay and 90% water. After air-drying this,
The dried product was mixed and pulverized to prepare a wettable powder.

Formulation Example 2 (Wettable powder) 1 ml of a mixture of 9% lactose, 1% zeolite and 90% water
Cell (JT-1211) 10 ⁹The individual was suspended. This was air dried
Thereafter, the dried product was mixed and pulverized to prepare a wettable powder.

Formulation Example 3 (Wettable powder) Cells (JT-121) per 1 g of a mixture of 15% diatomaceous earth, 77% kaolin, and 8% polyoxyethylene alkylphenyl ether
3) 10 ⁹ were suspended. After air-drying, the dried product was mixed and pulverized to prepare a wettable powder.

Formulation Example 4 (Wettable powder) diatomaceous earth 33%, carboxymethylcellulose 0.33%, water 6
6.67% of the mixture 1ml per cell were suspended (JT-1201) 10 ⁹ pieces of. After air-drying, the dried product was mixed and pulverized to prepare a wettable powder.

[0059] Formulation Example 5 (Dusts) hydroxypropyl -β- cyclodextrin 14%, were suspended white carbon 12%, clay 74% of the mixture 1g per cell (JT-1209) 10 ⁹ pieces of. After air-drying, the dried product was mixed and pulverized to prepare a wettable powder.

Formulation Example 6 (Granules) 10 ⁹ cells (JT-1165) per 1 g of a mixture of 15% β-cyclodextrin, 2% starch, 18% bentonite, 36% calcium carbonate, and 29% water After kneading, the mixture was granulated with a granulator and dried to prepare granules.

Formulation Example 7 (emulsion) Polyoxyethylene nonyl phenyl ether 18% ammonium phosphate, 6% polyoxyethylene nonyl phenyl ether, 29% triethyl phosphate, 47% tributyl phosphate
% Of the mixture cells per 1g (JT-1165) 10 ⁹ were then added to the uniformly suspended, it was prepared an emulsion.

[0062] Formulation Example 8 (oil) Spindle oil 95%, castor oil 4% silicone oil 1% mixture 1ml per cell (JT-1211) 10 ⁹ cells were uniformly suspended by adding, to prepare an oil. Formulation Example 9 (Dry flowable agent) Cells (J) per 1 ml of a mixture of sodium alkylbenzene sulfonate 12% and polyethylene glycol ether 88%
T-1209) 10 ⁹ were suspended to prepare a dry flowable preparation.

Formulation Example 10 (capsules) 0.7% sodium alginate, 5% kaolin, glycerin
10 ⁹ cells per ml of a mixture of 15% and 79.3% water (JT-1213)
The cells were suspended and dropped into a 0.2 molar calcium acetate solution to obtain a capsule-like product. After chopping this, it was sieved and air-dried to prepare a capsule.

Formulation Example 11 (capsule) 0.7% sodium alginate, 5% diatomaceous earth, 15 glycerin
%, Water 79.3% of the mixture 1ml per cell (JT-1165) 10 ⁹ cells were suspended to give a capsule form product was dropped into 0.2 molar calcium acetate solution. After shredding this, sieving,
It was air-dried to prepare a capsule.

[0065]

Industrial Applicability The present invention provides novel yeasts having a controlling effect on plant diseases. These yeasts can effectively control plant diseases with a smaller application amount than conventionally known microorganisms.

[0066]

[Sequence List] SEQUENCE LISTING <110> JAPAN TOBACCO INC. <120> NOVEL YEASTS, PLANT DISEASE CONTROL AGENT AND CONTROL METHOD USING THE YEASTS <130> J00-0084 <160> 4 <170> PatentIn Ver. 2.0 <210> 1 <211> 571 <212> DNA <213> Aureobasidium sp. <400> 1 aaaccaacag ggattgccct agtaacggcg agtgaagcgg caacagctca aatttgaaag 60 ctagccttcg ggttcgcatt gtaatttgta gaggatgctt tgggtaaaac gccagtctaa 120 gttccttgga acaggacgtc atagagggtg agaatcccgt atgtgactgg aaatgttaac 180 ctatgtaaag ctccttcgac gagtcgagtt gtttgggaat gcagctctaa atgggaggta 240 aatttcttct aaagctaaat attggcgaga gaccgatagc gcacaagtag agtgatcgaa 300 agatgaaaag cactttggaa agagagttaa aaagcacgtg aaattgttga aagggaagcg 360 cttgcaatca gacttgttta aactgttcgg ccggtcttct gaccggttta ctcagtttgg 420 acaggccagc atcagtttcg gcggccggat aaaggctctg ggaatgtggc ctccacttcg 480 gtggaggtgt tatagcccag tgtgtaatac ggccagccgg gactgaggtc cgcgcttcgg 540 ctaggatgct ggcgtaatgg ttgtaagcga c 571 <210> 2 <211> 560 <212> DNA <213 > unknown <400> 2 aaaccaacag ggattgccct agtaacggcg agtgaagcgg caacagctca aatttgaaat 60 ctggcttcgg cccgagttgt aatttgtaga ggatgctttt ggtgcggtgc cttctgagtt 120 ccctggaacg ggacgcctta gagggtgaga gccccgtaaa gttggacacc aagcctttgt 180 aaagctcctt cgacgagtcg agtagtttgg gaatgctgct caaaatggga ggtaaatttc 240 ttctaaagct aaataccggc cagagaccga tagcgcacaa gtagagtgat cgaaagatga 300 aaagcacttt gaaaagaggg ttaaatagta cgtgaaattg ttgaaaggga agcgctcatg 360 accagacttg tgccctgcgg atcatccagc cttctggctg gtgcacttcg cttggttcag 420 gccagcatcg gttctctgag ggggataaaa gcttcaggaa cgtggctcct ccgggagcgt 480 tatagcctgt tgcagaatac ccctcggggg accgaggttc gcgcatctgc aaggatgctg 540 gcgtaatggt catcagcgac 560 <210> 3 <211> 571 <212> DNA <213> Aureobasidium sp. <400> 3 aaaccaacag ggattgccct agtaacggcg agtgaagcgg caacagctca aatttgaaag 60 ctagccttcg ggttcgcatt gtaatttgta gaggatgatt tggggaagcc gcctgtctaa 120 gttccttgga acaggacgtc atagagggtg agaatcccgt atgtgacagg aaatggcacc 180 ctatgtaaat ctccttcgac gagtcgagtt gtttgggaat gcagctctaa atgggaggta 240 aatttcttct aaagctaaat attggcgaga gaccg atagc gcacaagtag agtgatcgaa 300 agatgaaaag cactttggaa agagagttaa aaagcacgtg aaattgttga aagggaagcg 360 cttgcaatca gacttgttta aactgttcgg ccggtcttct gaccggttta ctcagcttgg 420 acaggccagc atcagtttcg gcggccggat aaaggctctg ggaatgtggc ctccacttcg 480 gtggaggtgt tatagcccag ggtgtaatac ggccagccgg gactgaggtc cgcgcttcgg 540 ctaggatgct ggcgtaatgg ttgtaagcga c 571 <210> 4 <211> 571 <212> DNA < 213> Aureobasidium sp. <400> 4 aaaccaacag ggattgccct agtaacggcg agtgaagcgg caacagctca aatttgaaag 60 ctagccttcg ggttcgcatt gtaatttgta gaggatgatt tggggaagcc gcctgtctaa 120 gttccttgga acaggacgtc atagagggtg agaatcccgt atgtgacagg aaatggcacc 180 ctatgtaaat ctccttcgac gagtcgagtt gtttgggaat gcagctctaa atgggaggta 240 aatttcttct aaagctaaat attggcgaga gaccgatagc gcacaagtag agtgatcgaa 300 agatgaaaag cactttggaa agagagttaa aaagcacgtg aaattgttga aagggaagcg 360 cttgcaatca gacttgttta aactgttcgg ccggtcttct gaccggttta ctcagtttgg 420 acaggccagc atcagtttcg gcggccggat aaaggctctg ggaatgtggc ctccacttcg 480 gtggaggtgt tatagcccag ggtgtaat ac ggccagccgg gactgaggtc cgcgcttcgg 540 ctaggatgct ggcgtaatgg ttgtaagcga c 571

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B024 AA07 BA80 CA03 CA09 DA12 GA19 4B065 AA72X AA72Y BA22 CA47 4H011 AA01 BA01 BB21 BC03 BC06 BC07 BC08 BC16 BC17 BC18 BC19 BC20 BC22 DA05 DA13 DA15 DC03 DC05 DC08 DH02 DH03 DH03DH10DH

Claims (4)

[Claims] 1. A yeast exhibiting a controlling effect on plant diseases, wherein the base sequence of the D1 / D2 region of 26S rDNA is any one of the following base sequences (1) to (4): Kind. (1) A nucleotide sequence showing 99% or more homology with the nucleotide sequence described in SEQ ID NO: 1 (2) A nucleotide sequence showing 94% or more homology with the nucleotide sequence described in SEQ ID NO: 2 (3) A nucleotide sequence shown in SEQ ID NO: 3 A nucleotide sequence showing 98% or more homology with the nucleotide sequence (4) A nucleotide sequence showing 98% or more homology with the nucleotide sequence described in SEQ ID NO: 4 2. The yeast according to claim 1, wherein the nucleotide sequence of the D1 / D2 region of 26S rDNA is any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. 3. A plant disease controlling agent comprising the yeast according to claim 1 or 2 as an active ingredient. 4. A method for controlling plant diseases using the yeast according to claim 1 or 2.