JPS63198987A - Fusaric acid-decomposing or detoxicating gene - Google Patents

Abstract

PURPOSE:To facilitate prevention of disease participated by fusaric acid, by introducing the titled gene derived from fusaric acid-decomposed or fusaric acid-detoxicated microorganism into a microorganism or plant. CONSTITUTION:A microbial cell such as Cladosporium werneckii DK 2011 (FERM 8633), Pseudomonas cepacia UK-1 (FERM 9161), Pseudomonas solanacerum N50A16 (FERM 8841), etc., is ground to provide various kinds of DNA fragments, which are cloned into a vector pNo 1523 to prepare a gene library, which is introduced into Escherichia coli HB 101 and a strain having the strongest fusaric acid-detoxicating ability (PFS 4/HB 101) is selected. Plasmid PFS 4 is isolated from the strain and introduced into a microorganism capable of growing in a plant duct or growing in antagonism with the genus Fusarium or into a plant and can also be used for prevention of a disease participated by fusaric acid.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a fusaric acid degrading or fusaric acid detoxifying gene derived from a microorganism having the ability to decompose or detoxify fusaric acid.

(Prior art) Diseases that cause growing plants to wilt and die, such as 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 genus bacteria. The Fusarium genus bacteria that cause such infectious diseases include Fusarium oxysporum (Fusarium n+o) + Fusarium moniliforme (Fusarium n+o)
There are many types of bacteria such as niliform. These bacteria are soil contaminants, are absorbed from the soil into the plant body, and are transferred through the ducts. 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 has harmful effects not only on host plants but also on individual plants in the pond. Fusaric acid increases the permeability of plant plasma membranes.

As a result, Ca'', K''+N are present on the surface of the plant such as the second leaf.
This leads to the exudation of interstitial fluid containing cations such as ``a'' and various amino acids. When this dries, the osmotic pressure on the surface of the plant becomes high, which further increases water transpiration.

As a result, the plants will wilt and die.

As a drug to prevent Fusarium infections.

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

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; it must be mixed with the soil before planting Ti species or plants. .

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 CI> is metabolized and decomposed, and N
-Methylfusaric acid amide (I[). Furthermore, this fusaric acid decomposition effect is greater in cultivars with strong fusarium resistance.

Ctl+ (I) (II) Thus, if fusaric acid is rendered nontoxic through decomposition or chemical modification, it is thought that dehydration symptoms in plants due to water metabolism disorders can be prevented. For example, if microorganisms that can decompose or detoxify fusaric acid are obtained, it is thought that infectious diseases caused by Fusarium bacteria can be avoided. moreover,
If a gene related to fusaric acid decomposition or fusaric acid detoxification can be obtained from such a microorganism, a microorganism can be obtained in which the above-mentioned characteristics of the microorganism are enhanced.

It is thought to be useful for controlling the above plant diseases. Furthermore, it seems possible to introduce the gene into plants and create plants that are not affected by diseases associated with fusaric acid.

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

The aim is to provide genes involved in microbial-derived fusaric acid decomposition or fusaric acid detoxification that are effective in preventing or inhibiting plant diseases such as vine rot and wilt caused by Fusarium bacteria. There is a particular thing.

(Means and effects for solving the problems) The fusaric acid degrading or fusaric acid detoxifying gene of the present invention is derived from a microorganism having the ability to decompose or detoxify fusaric acid. These microorganisms are microorganisms that can grow in a medium containing fusaric acid and can decompose fusaric acid or render it nontoxic to plants. and bacteria have been found. For example, Talatosporium (C1ado-:
) microorganisms belonging to the genus Pseudomonas (Pseud
microorganisms belonging to the genus Omonas). What is a microorganism that belongs to the genus Thallatosborium?

Cladosporium werneckii (C1ados o
rumwerneckii) + especially Thallatosborium werneckii strain 0K2011 (Huikoken Bacterium No. 8
No. 633) is suitable. As a microorganism belonging to the genus Pseudomonas, Pseudomonas cepacia (Pseud
omonas7) lEspecially Pseudomonas cepacia t
The lK-1 strain (Feikoken Bibori No. 9161) was further developed by Pseudomonas solanacearum (Pseudomonas solanacearum).
Pseudomonas solanacearum belongs to the genus Pseudomonas solanacearum and is obtained by mutating the non-pathogenic Pseudomonas solanacearum.

Especially Pseudomonas solanacearum N50A16 strain (
8841) is suitable.

A typical example of microorganisms that have the ability to detoxify or detoxify fusaric acid is Taladosborium werneckii D.
The mycological properties, culture conditions, etc. of the K2011 strain, Pseudomonas cepacia UK-1 strain, and Pseudomonas solanacearum N50A16 strain are shown below.

(1) Koosboum Werne Key DK2011
This strain was isolated from soil by the inventors.

(1)-■1 Organized constituency This is an incomplete filamentous fungus consisting of osteoblasts that mainly form yeast-like colonies and hyphae that mainly form villous colonies.

Conidia formation is budding type, and the size of the conidia is 2 to 4 ×
It is 4 to 8 μm. The hyphae have septa and the width of the hyphae is 2.9
~5.8 μm.

(1)-■ Note: The culture medium does not need to be special. potassium, sodium,
Phosphates and sulfates such as magnesium.

Fusaric acid and various carbohydrates are added as a carbon source to a minimal medium containing chloride, etc. 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. Nitrogen sources used in this minimal medium include fusaric acid, inorganic nitrogen such as nitrates and ammonium salts, and organic nitrogen of amino groups. It is also possible to add cornstarch liquor, yeast extract, etc. as a nutritional source, if necessary. The culture temperature is 15°C to 35°C, preferably around 25°C. The culture pH is 6 to 9, preferably around 7, and the culture is carried out for 3 days to 1 week with aerobic stirring or shaking. Culture on a solid medium (agar medium) is also possible.

(1)-■7-q By growing the IJ% Dobuta microorganism in the above-mentioned suitable medium and culturing it aerobically in a normal medium containing fusaric acid, the fusarin contained in the medium can be removed. The acid is almost completely decomposed.

(2) Pseudomos cepacia UK-1 This strain was isolated from soil by the inventors.

(2)-■Remains] Dan 1 Kasumi It is shown in Table 1 below.

(Left below) Cladosporium Werneckii DK
A medium similar to that for the 2011 strain is used. The culture temperature is 15-40°C, preferably 25-30°C.

Culture pH is 4.0-9. The sail is preferably 5.5 to 6.5.

Culture is carried out for 1 to 5 days with aerobic stirring or shaking. Culture on a solid medium (agar medium) is also possible.

(2) -〇-Z The J'' L Needle Ice Shibamoto microorganisms are grown in the above-mentioned appropriate medium, and by aerobically cultivating this in a normal medium containing fusaric acid, The fusaric acid produced is almost completely decomposed.

(3) Shoot moss Solanacearum N50816 Bacteria of the genus Pseudomonas solanacearum are generally known as pathogens of bacterial wilt of Solanaceae plants and damping-off of tobacco, but non-pathogenic bacteria are also known. Adelberg's method using N-methyl-N'-nitro-N-nitrosoguanidine (NTG) to treat this non-pathogenic bacterium.
g) the method of et al. (Biochem.

Biophys, Res, Comm,, lfL,
788 (1965)), a non-pathogenic Pseudomonas solanacearum strain N50A16 having the ability to detoxify fusaric acid was obtained.

(3)-■ Plate Blood 1 The mycological properties of Pseudomonas solanacearum N50A16 strain are shown in Table 2 below.

(3)-■ The E-sho medium does not need to be special. potassium, sodium,
Phosphates and sulfates such as magnesium.

Various carbohydrates such as glucose and sucrose, casamino acids, and the like are added as carbon sources to a minimal medium containing chloride and the like. Nitrogen sources used in this minimal medium include inorganic nitrogen such as nitrates and ammonium salts, and organic nitrogen such as amino groups. Cornstarch liquor as a nutritional source,
It is also possible to add yeast extract or the like as necessary. The culture temperature is 10 to 40°C, preferably around 26°C. Culture pH is 4-8. The temperature is preferably around 6.5, and the culture is carried out for 3 to 7 days with aerobic stirring or shaking. Culture on a solid medium (agar medium) is also possible.

(3)-〇 Fusarin Festival When this microorganism is grown in the above-mentioned suitable medium and the obtained bacterial cells and/or its culture solution are brought into contact with fusarin acid,
Fusaric acid makes plants non-toxic (here, it refers to eliminating or reducing toxicity to plants). The ability of this microorganism to detoxify fusaric acid is
It is thought that it originates from the decomposition and/or chemical modification of fusaric acid.

The fusaric acid degrading or fusaric acid detoxifying gene of the present invention can be obtained by isolating DNA from a microorganism having the above-mentioned fusaric acid degrading or fusaric acid detoxifying ability by a conventional method, and
It can be obtained by extracting the gene involved in fusaric acid decomposition or fusaric acid detoxification from NA. Usually, the above bacterial cells are crushed.

DNA fragments are obtained by cleaving chromosomal DNA using various restriction enzymes. In some cases, DNA fragments are obtained by physically cutting during the DNA isolation process. For example, the above Talatosborium Wellnet-1--DK2
The bacterial cells of strain 011 were disrupted, and the method of Johnston et al. (1,
L, Johnston, S, G, Hughes
and^, J, CIutterbuckHHMB
O,J, 4, 1307-1311 (1985)
), various physically cut DNA fragments are obtained. For example, the known vector pN0152
3 (a part of pBR322 plus a streptomycin-sensitive gene) to create a gene library. Next, this is treated with a known bacterium 1, for example, 1 g of CaC, to obtain foreign DNA.
Host-organism with the ability to take in Escherichia coli HBOI (Escherichia parallel 11810
1) Transfer the transformant into a fusaric acid-containing medium, and select fusaric acid-resistant bacteria that can grow there. By culturing this fusaric acid-resistant bacteria in a fusaric acid-containing medium and examining the toxicity of the culture solution to plants, it can be confirmed whether the fusaric acid-resistant bacteria are capable of decomposing or detoxifying fusaric acid. Ru.

From the thus obtained fusaric acid-degrading or non-toxic surface (transformant), a plasmid is taken out by the Alka U-SDS method, etc., and the fusaric acid-degrading or fusaric acid-detoxifying gene on the plasmid is cut with various restriction enzymes. The location of is estimated.

By the above method, pNO1 cut at the Sma 1 site was
A plasmid obtained by cloning the above DNA fragment into 523 (the Sma I site disappears.
a 1 site is indicated by (Sm). ) was introduced into the strain with the strongest ability to detoxify fusaric acid (
pFS4/HBIOI) was selected and the plasmid (pFS4/HBIOI) was selected.
FS4) We conducted a study on the following. Plasmid pF
Restriction enzyme maps obtained by cutting S4 with various restriction enzymes are shown in FIGS. 1(a) to (e). FIG. 1(a) shows plasmid pFS4, and FIG. 1(a') shows an enlarged view of its foreign part. Diagonal lines indicate genes derived from vector pBR322. (middle) to (81) are DNA fragments cut with each restriction enzyme shown in FIG.

The host bacterium f! , E. coli HBIOI is resistant to streptomycin2, but if only vector plasmid pN01523 is introduced into it, pNO
The streptomycin susceptibility gene encoded on 1523 has come to exist in the country, resulting in dominant streptomycin sensitivity. Here, plasmid p
FS4 is the only Sma present in the streptomycin sensitivity gene of the above plasmid (pNO1523).
Since the r site is cleaved and foreign DNA is inserted there, the streptomycin sensitivity gene is inactivated. Therefore, the pFs4/HBIOI strain becomes streptomycin resistant. Since the ampicillin resistance gene in other regions on this plasmid pFSJ is kept alive, the pFs4/H8101 strain is resistant to both streptomycin and ampicillin.

The gene thus obtained can be introduced into microorganisms other than E. coli. For example, microorganisms that can grow within plants, preferably within the ducts of plants, and microorganisms that can grow competitively with Fusarium bacteria are suitable. Microorganisms that can grow within the ducts of plants include the non-pathogenic Pseudomonas solanacearum. Microorganisms that can grow competitively with Fusarium genus include bacteria belonging to Streptomyces (preferably 5 tretomc
essp, ATCC 39434), bacteria belonging to the genus Serratia (preferably Serratia marces)
cens) + Bacteria belonging to the genus Pseudomonas (preferably Pseudomonas fluo-L) - Pseudomonas
9 μKD-) + bacteria belonging to the genus Bacillus (Bacillus
ssubtilis).

Diseases associated with fusaric acid can be controlled by using microorganisms into which the gene of the present invention has been introduced. For example, two methods in which the gene of the present invention has been introduced and a microorganism that can grow in the ducts of a plant are absorbed by cutting a part of m (root wound inoculation method); Injecting into the detached base of the plant. Method: Cut the seedlings and techniques.

Fusarium disease can be avoided by injecting it into the plant by absorbing it through the cut (rooting after absorption to obtain seedlings). In addition, 2 Fusarium antagonistic bacteria with introduced genes
For example, diseases associated with fusaric acid can be effectively controlled by mixing it with soil or coating it on plant seeds.

Furthermore, it is also possible to obtain Fusarium disease-resistant plants by introducing the gene of the present invention into plants.

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

Back finger ■Top (A) Preparation of DNA: Cladosporium wernecki DK2011 strain was grown in PYE medium (peptone 0.3%
.

21 (containing 0.3% yeast extract) was cultured in an Erlenmeyer flask at 30°C for 3 days. The culture solution was centrifuged, and the precipitated bacterial cells were mixed with 100++M EDTA (pH 8,
5). After freezing this at -80℃, 3
The operation of melting at 0°C was repeated three times. Grind the bacterial cells with a homogenizer and centrifuge (10,000xg,
for 15 minutes). Add 0.2% SOS to the precipitate.

Tris-HCI 5h+M, EDTA 100mM
, and a solution containing NaCl at a ratio of 50 μβM (
pH 7,8) at 10% per 2g (wet) of the above bacterial cells.
- added at a rate of -. This was incubated at 68 °C for 15 min and centrifuged (10,000 x g.

RNase^ (manufactured by Sigma, preheated at 100°C for 15 minutes), and the resulting supernatant was treated with DNA.
to inactivate se) was added to a final concentration of 100 μg/−. This was incubated at 30°C for 20 minutes and treated with proteinase K (Boehringer).
was added to give a final concentration of 50 μg7yd. 37
After incubation for 20 minutes at °C and phenol treatment twice, an equal volume of chloroform-isoamyl alcohol mixture (24:1) was added to the aqueous layer. This is centrifuged (
8000 x g for 10 min), and 1710 volumes of 3M sodium acetate solution and 3 volumes of cold ethanol were added to the supernatant. This was then kept at -70°C for 20 minutes and the DN
A was precipitated. This was centrifuged (10,000 x g
After removing ethanol from the precipitate (DNA) by drying (for 15 minutes), it was dissolved in sterile water at a concentration of 0.5 μg/μβ. In this way, a solution containing about 1 μm of DNA was obtained. It was confirmed by electrophoresis that this DNA was physically cut during the above series of operations.

([1) Cloning (B) - ■ Cleavage of pNO1523 at the S+++a I site: As a vector, pNO1523 (Pharma
Made by cia: Vector HD, Dern, which has a streptomycin sensitivity gene added to a part of pBR322.
Gene+ 15+ 99-102+ (1981)
)It was used. pN01523 (0.3μg/μJ)
40pi, Sma I (5 yt/p I
I; Manufactured by N1ppon Gene) 2μf, Small
A mixture of 5 μl of the 10-fold concentration reaction solution and 3 μl of sterile water was incubated at 30° C. for 1 hour. After phenol treatment and ethanol precipitation, the precipitate was treated with Tris-E.
It was dissolved in 40 μl of DTA (Tl buffer). 10 μβ of it was subjected to electrophoresis, and it was confirmed that pNO1523 was cleaved at the Sma I site.

(B)-〇 Ligation: S obtained in this way
5 μf of solution containing pNO1523 cleaved at ma 1 site, DNA obtained in section (A) (5 μg/μl)
3 μJ, T, ligase (300 units/μl) 1
μl.

10x reaction solution for T4 ligase (500mM) Lith-hydrochloric acid (pH 7,9), 100mM MgCl□200m
(contains M dithiothreidole) 2N 1.10mM
Mix 1 ATP2 tt and 7 μl of sterile water and incubate at 16°C.
I let it react overnight.

(C) Transformation: The reaction solution obtained in section (B) was transformed into Ca
The cells were brought into contact with C1z-treated E. coli (IJIW IBIOI) and transformed. This was then treated with streptomycin (
100μg/d) and ampicillin (50μg/-)
The cells were spread on a L Broth agar medium containing L Broth, and cultured at 37°C for 24 hours. The details of the preparation of CaClg-treated bacteria and the above transformation method were according to the method of Ikeda et al. (Genetics Experimental Method Course 1.13H1983; Kyoritsu Shuppan; edited by Haruo Koseki and Reibe Shimura).

(D) Screening for fusaric acid detoxification genes: (C
Approximately 500 bacteria obtained in section 2) were plated on L Broth agar medium containing 50 μg of fusaric acid on ground, and bacteria that conferred resistance to fusaric acid were selected. As a result, a transformant exhibiting resistance to fusaric acid was obtained. Among them, one transformant showing high resistance was selected. This was cultured, and a plasmid was obtained from the cells using the alkaline-8DS method. When this plasmid was reintroduced into E. coli strain 08101, it was confirmed that it had a strong ability to detoxify fusaric acid. This E. coli plasmid was named pFS4,
The above transformant into which pFs4 was introduced was transformed into Escherichia
Cori (Escherichia nandan) pFs4/H
It was named BIOI strain (Feikoken Bibori No. 9161). It was confirmed that this transformant had the same mycological properties as the parent strain, except that fusaric acid was rendered nontoxic.

E. coli pF, the transformant obtained in Example (D)
The S4/IIBIOI strain was cultured in L Broth medium at 37°C.

The obtained bacteria were suspended in physiological saline at a density of 10' cells/d. Separately vector plasmid pN01
E. coli HBIOI into which only 523 was introduced (pN01
523/IIBIOI strain) was prepared according to the method of Example 1, and a suspension of 10' cells/- in physiological saline was similarly prepared. Five tomato cuttings at the 5th to 6th node stage were used, and the cut ends were immersed in each of the above bacterial suspensions and physiological saline for 3 hours. After washing the cut portion with water, it was immersed in about 15 d of a 100 μg/− fusaric acid aqueous solution and allowed to absorb for 40 hours. Water was added to compensate for the amount lost due to absorption and transpiration of the fusaric acid aqueous solution, and the temperature was always maintained at about 15 d.

When the above water and the pNO1523/HBIOI strain were absorbed, withering from the stems was observed. However, the above pF
When the s4/HBIOI strain was absorbed, no abnormality was observed in any of the five plants.

Back coating pPs4/HBIOI strain obtained in Example 1 was
L Broth medium containing g/- of fusaric acid was inoculated.

Shaking culture was performed at 30°C. 0.24.48.72
After a period of time, a portion of the solution was taken and centrifuged, and the supernatant was collected.

This was then filtered through a 0.2 μm Millipore filter. The obtained filtrate and MS medium (Murashige
-Skoog medium: Physiol, Plant,
15.473 (1962)) at a volume ratio of 1:4. Add tomatoes (L co ersicon) to this.
esculenfum Mill cv, Zuik
Free single cells of the callus induced from the cambium part of the stem of (o) were added at a ratio of 10' to 10' cells/IR1 and left at 25°C for 24 hours. After 24 hours, callus cells were fluorescently stained with fluorescein diacetate (PDA). This was visually observed using a fluorescence microscope, and the survival rate (%) of callus cells was calculated (n=500). Table 3 shows the tomato callus survival rate.

Table 3: Insert the blade of a utility knife perpendicularly into the vermiculite surface approximately 21 cm outward from the base of the stem of a tomato grown in a pot containing vermiculite (4th node development stage, approximately 30 days after sowing). The tomato root was cut to a depth of about 53° and about 51°. In this part, 10 m of the physiological saline suspension of the L dish pFS4/HBIOI strain obtained in Example 1 (
For comparison, instead of the above bacterial suspension, water 10- and pNO
1523/) 18101 strain suspension 10a1 was used in the same manner. Cultivation was continued for one week to allow the roots to recover. After removing the vermiculite and washing the roots with water, add μ to Fusarium qJ - f, sp, b
Spore suspension of kJ target rsi play (10'/mg) 50
- for several minutes to allow spores to adhere to the roots (root immersion inoculation method), and without washing, the plants were transferred to a new pot containing vermiculite and cultivation continued at 20°C. Separately, p
A tomato strain to which the Fs4/HBIOI strain suspension was not inoculated and to which the Fusarium spores were not attached was similarly cultivated.

The tomato strain inoculated with the above pPs4/HBIOI strain is 1
Even after several months, no abnormalities were observed, indicating the extent of the growth.

It was similar to the tomato strain to which the Fusarium genus bacteria was not attached. In contrast, water and pN01523/
In the tomato strain using the HBIOI strain, wilting symptoms appeared on the leaves from 8 days after contact with the Fusarium genus bacteria.

After one month, significant growth failure was observed, with severe withering of lower leaves and browning of the stems.

The number of stem nodes was small and the growth status was poor.

The pFS4 obtained in Example 1 was obtained by cutting and cutting the seedlings of standing tomato.
/ IIBIOI strain suspension (10 pieces/me) 10d
Soaked in for 3 hours. The cut sections were washed with water, immersed in water, and left at room temperature. Fusariu after adventitious roots are formed on the cut.
spore suspension (
10'/mff1) 50- for several minutes to allow the spores to adhere to the roots. Without washing, the plants were transferred to new pots containing vermiculite and cultivated at 20°C.

For comparison, the same operation was performed using an aqueous suspension pNO1523/IIBIOI strain suspension instead of the pFs4/HBIOI strain suspension. Separately, pFs
Tomato strains that were not immersed in the 4/HBIOI strain suspension and to which the water suspension Zalium genus bacteria were not attached were also cultivated in the same manner.

The tomato strains inoculated with the above pFS4/HBIOI strain are 2
Even after several months, no abnormalities were observed, indicating the extent of the growth.

It was similar to the tomato strain to which the Fusarium genus bacteria was not attached. In contrast, water and pN.

In the tomato strain using the 1523/HBIOI strain, wilting symptoms appeared on the leaves from 8 days after contact with the Fusarium genus bacteria. After one month, significant growth problems were observed, with severe curling of the lower leaves and browning of the stems.

The number of stem nodes was small and the growth status was poor.

Ura 1: Using strawberries instead of tomatoes, Fusariumy
The procedure was the same as in Example 4, except that Fusarium mf, sp, and Fusarium mf, sp, and Fusarium mf, sp, and 1 coersici were used instead of σ, μm-f, sp, and 1 coersici. Results similar to those in Example 4 were obtained.

(Effects of the Invention) According to the present invention, a fusaric acid degrading or fusaric acid detoxifying gene derived from a fusaric acid degrading or fusaric acid detoxifying microorganism can thus be obtained. Plant diseases associated with fusaric acid can be effectively controlled by using a transformed microorganism in which this gene is introduced, for example, into a known microorganism, or by introducing the gene into a plant.

4. Figure 1 is a restriction enzyme map of plasmid pFS4 containing two genes obtained according to the present invention.

Claims (1)

[Scope of Claims] 1. A fusaric acid degrading or fusaric acid detoxifying gene derived from a fusaric acid degrading or fusaric acid detoxifying microorganism. 2. The microorganism is Cladosporium
The gene according to claim 1, which belongs to the genus Sporium. 3. The microorganism is Cladosporium wernecki.
The gene according to claim 2, which is i\). 4. The gene according to claim 3, wherein the microorganism is Cladosporium werneckii strain DK2011 (Feikoken Bacterium No. 8633). 5. The microorganism is Pseudomonas
The gene according to claim 1, which belongs to the genus o-nas\). 6. The microorganism is Pseudomonas cepacia (¥Ps
The gene according to claim 6, which is eudomonas ¥ cepacia ¥). 7. The microorganism is Pseudomonas cepacia UK-1
The gene according to claim 5, which is a strain (Feikoken Bibori No. 9160). 8. The microorganism is Pseudomonas solanacearum (
￥Pseudomonas￥ ￥solanacear
um\) and is nonpathogenic. 9. The gene according to claim 8, wherein the microorganism is obtained by subjecting non-pathogenic Pseudomonas solanacearum to mutation treatment. 10. The gene according to claim 8 or 9, wherein the microorganism is Pseudomonas solanacearum N50A16 (Feikoken Bibori No. 8841) strain.