JPH0710901A - Polysaccharides m-100 substance group and production thereof - Google Patents

Abstract

PURPOSE:To heighten resistance to a plant pathogen by culturing a specific microorganism producing polysaccharides M-100 substance group. CONSTITUTION:A microorganism belonging to the genus Colletotrichum is inoculated into a medium containing a carbon source, a nitrogen source, an inorganic salt, etc., the microorganism produces polysaccharides M-100 substance group having the following physicochemical properties: mol.wt., 7,500-13,000; action, at least a chitinase induction activity; composition, 100% glycose; coloration reactions, positive in phenol/sulfuric acid reaction, negative in Lowry reaction, negative in Elson-Morgan reaction, negative in carbazole/sulfuric acid reaction, etc. The inoculated microorganism is cultured with aeration and agitation at 20-40 deg.C to obtain a culture solution, which is homogenized and then extracted with hot water. The extract is passed through a strongly basic anion exchange resin at a pH of 10 or higher to adsorb substances produced by the microorganism onto the resin. The absorbed substances are eluted with a salt and concentrated with an ultrafiltration membrane having a fractional mol.wt. of 3,000. The concentrate is collected and subjected to gel filtration to recover the fractions having mol.wt. of 7,500-13,000. Thus, the objective polysaccharides are obtained.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a molecular weight of 7500 to 13 which induces at least chitinase and hypersensitivity reaction which are resistance reactions against pathogens of plants such as tomato.
Related to the substance group of polysaccharide M-100 included in 000 and its production method.

[0002]

2. Description of the Related Art In order to prevent infection of plants with pathogens, fungicides are generally applied. On the other hand, the plant recognizes the invasion of the pathogen, synthesizes an antibacterial substance such as phytoalexin, and protects itself. Substances that induce such resistance reactions are elicitors (plant cell engineering, second
Vol., Supplement 1, p. 399, 1990). As a disease control method utilizing the resistance of the plant itself, a method of inducing resistance by pseudo-infecting a plant with a part of a pathogen is known (Kitanihon Pest Research Institute,
Volume 38, page 10, 1987) for attenuated viruses (plant protection, volume 43, number 1, page 11, 198).
It has not been put into practical use except for 9 years.

[0003]

Problems to be solved by the invention In general, the disease control by spraying pesticides has many safety-related problems such as the effects of pesticides remaining on crops on the human body and environmental pollution. Further, in terms of the effect of pesticides, there is a problem that once the pathogen invades the plant, the pesticide can no longer exert its bactericidal effect.

The polysaccharide component of the pathogen which can be recognized by the plant is isolated and sprayed on the plant to cause pseudo-infection, whereby the plant produces a resistance reaction to the pathogen.
Since the polysaccharide component is easily decomposed, there is no risk of contamination of the human body and environment. Furthermore, the polysaccharide acts on the plant as an information transmitter, and the plant cell recognizes this and synthesizes an antibacterial substance, and thus inhibits the growth of the pathogen invading the plant (Plant Cell Engineering, Volume 2, Supplement 1, 399, 1
990), the polysaccharide M-100 substance group of the present invention was newly searched based on such findings.

[0005]

Means for Solving the Problems The present invention is a novel substance, a polysaccharide M-100 substance group, which has an activity of inducing a resistance reaction which the plant itself has against a plant pathogen, such as choletotrichum. Fragarie M601
3 (FERM P-12625; Colletotrichumfragar
iae M6013), which is a bacterial cell component, is obtained by a general isolation method for β-1,3-glucan, and is inactivated by β-1,3-glucanase treatment, and has a molecular weight of at least a chitinase activity of 7500. To 13000, a method for producing a polysaccharide M-100 substance group using a microorganism belonging to the genus Choletotrichum and having the ability to produce the polysaccharide M-100 substance group. .

[0006] Microorganisms used in the present invention is a filamentous fungus that belongs to collect birds Kum genus capable of producing a polysaccharide M-100 substance groups, as a typical strain, for example, Colletotrichum & Furagarie (Colletot
richum fragariae ) M6013 is mentioned. This strain is Microorganism Research Institute No. 12625 (FERM P-1262).
5) has been deposited at the Institute of Microbial Technology, Institute of Industrial Technology. The mycological properties are shown below.・ Growth condition in each medium 1. In the case of Czapek agar plate (Cz) at 25 ° C, the growth is rather fast, and the size of the colony is 40-43 mm in 7 days and 60 mm or more in 14 days.
The flora is thick and the surface becomes fluffy due to aerial hyphae. The color of the colony is pale gray-grey (1-3B1). The perimeter is all around. Give out a colorless leachate. Does not emit diffusible dye. The back side is gray, green gray or dark green grey, greenish
gray or dark green (28-30F1-3). 2. In the case of wort agar plate (MA) at 25 ° C, the growth is rather fast, and the size of colonies is 40-45 mm in 7 days and 60 mm or more in 14 days.
The flora is slightly thicker and more finely fluffy than that of Czapek agar plate. Colony color is yellow gray y
ellowish gray (2BC-2). Give out a colorless leachate. Does not emit diffusible dye. Convex lens type. The surrounding area is almost full. The back side is yellowish gray-greyish yellow (2B2-
3). 3. In the case of potato-glucose agar plate (PAG) at 25 ° C, the growth is rather fast, and the size of colonies is 42-44 mm in 7 days and 60 mm or more in 14 days.
The surface changes from fluff to velvet due to aerial hyphae. Colony color is greenish gray-yel
lowish gray (1-3C2). Give out a colorless leachate. Does not emit diffusible dye. The back side is yellow gray.

Morphological characteristics under a microscope Conidia formation mode is a phialophore type, and a single conidia forming cell is directly generated from hyphae. Conidia forming cells are short and sparsely distributed. Conidia develop in the form of mucus at the tips of forming cells. Conidia are 12-22 × 4-6 μm in size. There are thin cylinders with rounded ends, thin ellipses and oval shapes, which are colorless and have smooth walls. Before germination, it is slightly brown and has one septum, but sometimes two septum. Brown and close to circular or irregular size 7-1
A 2 × 6-10 μm appressorium is formed directly from the hyphae, at the tip of the hyphae, or in the form of side branches. Neither conidia or associated bristles nor asci were formed. In addition, the color display of the growth state in each of the above-mentioned medium, Kornerup,
A. and Wanscher, JH 1978. “Methuen handbook of co
lour. 3rd ed. ”Eyre Methuen, based on the indication of London.

Physiological properties pH that can grow: 3.5-7.5 Optimum growth pH: 4.5-6.5 Temperature that can grow: 13-37 ° C Optimum growth temperature: 21-30 ° C

[0009] M6013 strains isolated from and identified strawberries, the form of conidia, Colletotrichum sp C from morphological characteristics, such as having appressorium
It was considered to be a microorganism belonging to olletotrichum . As a result of observing this strain in detail, “Tsutomu Yamamoto, a new disease of strawberry“ Anthracnose ”, Plant Protection Vol. 25, 61-64, 1971”
And "K. Okayama, Pathogens of Strawberry Anthracnose and Morphology, Plant Protection, Vol. 42, pp. 559-563, 1988" reported by Colletot.
richum fragariae ). Therefore, this strain is C.
thought to be fragariae . “Von Arx, JA Die Arten d
er Gattung Colletotrichum Corda. Phytopathol. Z. 2
9 : 413-468.1957 ”,“ von Arx.JA Plant Pathogenic
Fungi, J. CRAMER, Berlin. 288p, 1987 ”described the incomplete generation of the ascomycete, Glomerella cingulata , in C. gloeospo .
rioides and C. fragariae are treated as synonyms. On the other hand, “Howard, CM and Albregts, EE Bl
ack Leaf Spot Phase of Strawberry Anthracnose Caus
ed by Colletotr ichum gloeosporioides ( C. fragaria
e ), Plant disease.67: 1144-1146,1983 ”
C. gloeosporioides forms an ascospore in culture ( ascomycete fungus), while C. fragariae does not form ascomycete in culture (incomplete fungus). I am going to do it. As for this strain, the formation of ascitic shell was not observed. As described above, the morphological characteristics are the same, they do not form ascidian husks, and they are separated from strawberries.
“Howard, CM and Albregts, EE Black Leaf Spot Ph
ase of Strawberry Anthracnose Caused by Colletotri
chum gloeospirioides ( C. fragariae ). Plant diseas
e. 67 : 1144-1146,1983 ”, this strain was identified as Colletotrichum fragariae . However, when the formation of the ascitic shell was observed, C
Should be olletotrichum gloeosporioides .

The medium component for culturing the microorganism used in the present invention is not particularly limited as long as it can be used by the microorganism, but typical examples of the medium component are as follows. That is, the carbon source is starch, dextrin and glucose, preferably soluble starch. The nitrogen sources are casein and peptone, preferably yeast extract. The inorganic salts are phosphates and magnesium salts, preferably dipotassium phosphate and magnesium sulfate. An antifoaming agent can be added to the medium and is preferable. As the defoaming agent, for example, Disfoam CC-222 is preferably used. A typical medium composition of the medium of the present invention is as follows, for example, per liter of medium. Soluble starch 1.5-150 g, preferably 15 g yeast extract 0.5-50 g, preferably 5 g 1 potassium phosphate 0.1-10 g, preferably 1 g magnesium sulfate 0.05-5 g, preferably 0.5 g Disform CC-222 0.1-100 ml, Preferably 0.
4 ml This medium is adjusted to pH 6.0 with hydrochloric acid. A microorganism-containing culture solution can be obtained by culturing a microorganism in such a medium at 20 to 40 ° C., preferably 30 ° C. for 5 days with aeration and stirring. The polysaccharide M-100 substance group of the present invention can be separated and purified from the bacterial cells thus obtained, for example, by a general method for isolating and purifying β-1,3-glucanase. . For example, it is separated and purified as follows. That is, the suspension of cells is homogenized, extracted with hot water, and adsorbed to a strongly basic anion exchange resin such as Dowex-1 at an alkaline pH of 10 or more to obtain 50 mM NaC.
Elute with a salt such as l. Furthermore, the molecular weight cutoff of 3000
The concentrated fractions by the ultrafiltration membrane of (1) were collected, and each component having a molecular weight of 7500 to 13000 was obtained by gel filtration to obtain a group of novel substances having at least a strong chitinase activity-inducing activity.

By contacting the polysaccharide M-100 substance group of the present invention with a plant, the resistance of the plant to pathogens can be increased. Polysaccharide M-10 of the present invention
The means for bringing the 0 substance group into contact with the plant is not particularly limited, and examples thereof include application to seeds, immersion of seeds in the substance solution, spraying on leaf surfaces, addition to cultivated soil, and liquid for hydroponics. It can be added to fertilizers and virus-free seedlings to the conditioned medium.

[0012]

EXAMPLES The present invention will be described more specifically by way of examples. The present invention is not limited to these examples. Example 1 Culture Production strain Choletotrichum fragalier M6013 (F
ERM P-12625 Colletotrichum fragariae M
6013) in a 500 ml Erlenmeyer flask into which 150 ml of a medium having a composition of soluble starch 15 g / liter, yeast extract 5 g / liter, potassium 1 phosphate 1 g / liter, magnesium sulfate 0.5 g / liter (pH 6.0) was dispensed. Inoculated with 10 ml / inoculum after culturing at 30 ℃ for 3 days
Lithium, Disfoam CC as an antifoaming agent for inoculum medium
-222 (manufactured by Nippon Oil & Fats Co., Ltd.), a 50 liter jar fermenter filled with 40 liter of a medium added with 0.2 ml / liter, aeration at 30 ° C., 11 liter / min, stirring 10
The cells were cultured under the condition of 0 rpm for 4 to 5 days and subjected to basket centrifugation to obtain 1.3 kg of bacterial cells.

Example 2 Preparation 1.3 kg of cells were treated twice with 4 liters of acetone and 2 times with 4 liters of chloroform-methanol (1: 1 mixture).
The cells are defatted by being treated once, suspended in 9 liters of distilled water, and added to a polytron homogenizer (KINEMATI).
After crushing with CA), heat extraction at 120 ° C for 30 minutes, 30
A crude extract was obtained by centrifugation at 00 rpm for 5 minutes. This crude extract was subjected to ultrafiltration (pore size 0.25 μm, polyolefin membrane manufactured by Asahi Kasei) to remove cloudy particles, and glycine was added to 10 mM to adjust pH to 8 with NaOH, and then 20 mM glycine-NaOH buffer (pH 8.0). Equilibrated with Dowe
Unnecessary components such as proteins were removed by passing through an x-1 column (inner diameter 44 mm). NaOH is added to this passing liquid.
Was added to adjust the pH to 10.5, and 20 mM glycine-NaO was added.
The active ingredient was adsorbed on a Dowex-1 column (inner diameter 44 mm) equilibrated with H buffer (pH 10.5), washed with water and then 5
Fractions 2 to 4 eluted with 0 mM NaCl (1 fraction 9999
88 mg of active fractions obtained by desalting and concentrating the drops) by ultrafiltration (pore size 0.25μ, polyolefin membrane manufactured by Asahi Kasei) to remove turbid particles, and further ultrafiltration (molecular weight cutoff 3000, polyacrylonitrile membrane manufactured by Asahi Kasei). Obtained. Fraction N of Bio-Gel A-5m (column inner diameter 32 mm, 1 fraction 1000 drops) (FIG. 3) to isolate the active ingredient from the active fraction
o. 25 to 30 were collected and further subjected to gel filtration using Bio-Gel P-30 (column inner diameter 32 mm, 1 fraction 250 drops) (FIG. 4) to fraction No. 8, No. 9 and No.
10 were collected. First, this fraction No. Polysaccharide having a molecular weight of about 7650 after drying under reduced pressure from 10 (polysaccharide M
-100-1) 15 mg was obtained, then fraction no.
22 mg of a polysaccharide having a molecular weight of about 10,000 (polysaccharide M-100-2) was obtained from 22 by drying under reduced pressure. After drying under reduced pressure from No. 8, 32 mg of a polysaccharide having a molecular weight of about 13,000 (polysaccharide M-100-3) was obtained. These polysaccharides have the following physicochemical properties, and are polysaccharides M-100, which are peak components showing strong chitinase activity-inducing activity.
Obtained as a substance group. The total sugar content of each fraction was measured by the phenol-sulfuric acid method, and the activity of each peak was measured by the method described in Example 3.

Physicochemical properties Included in the molecular weight: 7500-13000. Measured by gel filtration high performance liquid chromatography with Asahipak GS-510 (HPLC, mobile phase: water, molecular weight standard: pullulan (Showa Denko)). Retention time of each peak component in the above HPLC Fraction No. 8: Molecular weight around 13,000: retention time 16.00 minutes. Fraction No. 9: Molecular weight around 10,000: retention time 16.43 minutes. Fraction No. 10: Molecular weight around 7650: retention time 16.87 minutes. Constituent sugar: Glucose is 100% in each of the polysaccharide M-100 substance groups from each fraction. Determined by performing hydrolysis under conditions of 1.2N HCl, 80 ° C., 24 hours, and measuring free glucose by high performance liquid chromatography using TOSOH Amide-80 (mobile phase: acetonitrile-water 60:40). Color reaction Phenol-sulfuric acid reaction: Positive Lowry reaction: Negative Elson-Morgan reaction: Negative Carbazole sulfuric acid reaction: Negative Solubility water: Soluble ethanol: Insoluble Acetone: Insoluble Chloroform: Insoluble Benzene: Insoluble Acid, neutral, basic : Neutral substance color: White IR absorption spectrum: Figure 1. (KBr) 3400, 2950, 1670, 1
Absorption at each wave number near 560, 1400 and 1350 cm ^-1 . UV absorption spectrum: Figure 2. No particularly characteristic peak (water: 1 mg / ml)

Example 3 Action The action of the polysaccharide M-100 substance group of the present invention was measured using chitinase as an index of the defense reaction of plants against infection by pathogens. That is, tomato seeds (variety: Momotaro)
On a cotton wool containing distilled water at 26 ° C. for 14 days, the resulting seedlings are immersed in an aqueous solution of each component of the polysaccharide M-100 substance group, and 18 hours later, the seedling extract is prepared by the following method. Chitinase activity was measured. As a result, FIG. 5 (polysaccharide M-100-1) and FIG. 6 (polysaccharide M-100-2)
And as shown in FIG. 7 (polysaccharide M-100-3), the polysaccharide M-10 in the substance group of the polysaccharide M-100 of the present invention.
0-1 is 2 μg / ml or more, polysaccharide M-100-2 is 4 μg
/ Ml or more, and the polysaccharide M-100-3 was found to induce the chitinase activity of tomato seedlings at a concentration of 10 μg / ml or more. The activity was measured as follows. That is, after washing the seedlings with water, water on the surface of the seedlings is removed,
After freezing at 100 ℃ for 1 hour, 4 times amount of seedling weight (g) in acetate buffer (0.2M, pH5.2, 2.5% polyvinylpyrrolidone, molecular weight 44000, 0.5% polyethylene glycol, molecular weight) 6000, containing 0.01% β-mercaptoethanol), crushed in a mortar, and
Squeeze with heavy gauze, centrifuge at 12000 xg for 10 minutes,
The supernatant was used as a crude enzyme solution. To 0.1 ml of this crude enzyme solution, 1 ml of the above acetate buffer containing 0.05% ethylene glycol chitin (Sigma) was added, reacted at 28 ° C. for 15 minutes, and then 0.5 M containing 0.5 g / liter potassium ferricyanide. 2 ml of sodium carbonate solution was added, boiled for 15 minutes, cooled, and then OD 420 nm was measured. The activity that reduces the OD value by 0.1 per minute is defined as 1 mU. The activity is expressed in terms of 100 g of fresh weight of seedlings.

Example 4 Action As another indicator of the plant defense reaction induced by the polysaccharide M-100-1, cell killing is caused by overproduction of an antibacterial substance synthesized by some plant cells in an individual. The hypersensitive reaction that protects the individual by causing it was measured.
As a result, as shown in FIG. 8, it was confirmed that the polysaccharide M-100-1 induces a hypersensitive reaction at a concentration of 1 μg / ml or more. The activity was measured as follows. That is, 1 mg / liter of naphthalene acetic acid, 0.5 mg / liter of benzyladenine, and 0.5 mg / liter of 2,4-D (2,4) were obtained from the seedlings of tomato (variety: Momotaro).
-Dichlorphenoxyacetic acid), callus induction in a Rinsmeier-Skoog medium containing 0.7% agar, and the obtained callus was shake-cultured for 20 days in the same liquid medium except that it did not contain agar, and the culture solution was The polysaccharide M-1 obtained in Example 2 was added to 0.25 ml of a culture solution in which callus was uniformly dispersed by filtering through a nylon sieve (32 mesh) to remove large cell aggregates.
0.25 ml of a solution of 00-1 diluted with water was added,
After incubating for 3 hours, MTT (3- (4,5-d
imethylthiazol-2-yl) -2,5-diphenyl tetrazolium brom
ide) solution (0.05 ml) and further incubated at 30 ° C. for 2 hours, and then 0.5 ml of isopropyl alcohol containing 0.04N hydrochloric acid was added to extract the reaction product formazan produced in the cells, which was OD570 nm. It was measured at. This formazan production is an index of living cells, and the hypersensitive reaction shows 100% survival rate of untreated cells.
The relative survival rate was shown.

Example 5 Enzymatic Degradation In order to investigate the properties of this substance, various polysaccharides were degraded with hydrolases, and the chitinase-inducing effect of the reaction products on tomato seedlings was observed. Examples of sugar hydrolase include α-amylase (Seikagaku Corporation), cellulase TC
(Manufactured by Seikagaku Corporation) and Zymolyce 100T (manufactured by Seikagaku Corporation) containing β-1,3-glucanase were used. Using 100 μg / ml of the present polysaccharide M-100 substance as a substrate, the enzyme and the enzyme were added to 10 U / ml so as to obtain 10 U / ml.
Add heat-inactivated water in boiling water for 1 minute to adjust the pH to 6.0.
The reaction was carried out in 0 mM phosphate buffer at 45 ° C for 1 hour. The tomato seedlings were dipped in this reaction solution, and the chitinase-inducing effect of the tomato seedlings was observed according to the method described in Example 4. As a result, Table 1 (polysaccharide M-100-1),
Table 2 (polysaccharide M-100-2) and Table 3 (polysaccharide M-
As shown in 100-3), it was confirmed that all of the present polysaccharide M-100 substance groups were inactivated by the treatment with Zymolyce 100T containing β-1,3-glucanase.

Table 1. Chitinase Induction of Tomato Seedlings by Enzymatic Degradation of Polysaccharide M-100-1 Chitinase Activity (U / 100g) Addition Enzyme Reaction Product Reaction Product by Heat Inactivating Enzyme-33.7 36.4 α-Amylase 31.7 29.0 Cellulase TC 33.4 37.6 Zymolyce 100T 0 31.3

Table 2. Chitinase Induction of Tomato Seedlings by Enzymatic Degradation of Polysaccharide M-100-2 Chitinase Activity (U / 100g) Addition Enzyme Reaction Product Reaction Product by Heat-Deactivating Enzyme-28.9 28.1 α-Amylase 29.7 27.2 Cellulase TC 26.9 29.4 Zymolyce 100T 0 27.1

Table 3. Chitinase Induction of Tomato Seedlings by Enzymatic Degradation of Polysaccharide M-100-3 Chitinase Activity (U / 100g) Addition Enzyme Reaction Product Reaction Product by Heat-Deactivating Enzyme-31.5 32.3 α-Amylase 30.6 29.8 Cellulase TC 30.4 31.8 Zymolyce 100T 0 31.5

Example 6 Novelty Regarding polysaccharides having several glucose components,
Based on the method described in Example 3, the chitinase inducing effect was examined. As a result, as shown in Table 4, the polysaccharide M
Except for -100 substances, no chitinase-inducing effect was exhibited.

Table 4. Effect of various polysaccharides on induction of chitinase in tomato seedling Polysaccharide (100 μg / ml) Molecular weight binding chitinase activity (U / 100g) No addition 0 Pullulan R-5 (Showa Denko) 5,800 α-1,60 Amylose Ex-1 (raw Chemical industry) 2,900 α-1,40 Laminarin (Seikagaku) 1,153 β-1,3 0 Polysaccharide M-100-1 7,650 β-1,3 32.5

Example 7 Uses Seedlings obtained from a runner of a virus-free parent strain of strawberry (variety: Onamine) were treated with PLUS ONE (green compost, C / C).
N = 6.6, NPK = 1.4-0.6-0.06%) 150g / 10l, Yogurus No. 2 (Toyo Brewing Organic Material, NPK = 5-
2-3%) 15 g / 10 liters, strawberry mixed fertilizer (Shizuoka Prefecture Economic and Agricultural Cooperative Federation, synthetic fertilizer, NPK = 6-7-6%) 1
3.5 g / 10 liters, long 140 (slow-acting chemical fertilizer manufactured by Asahi Kasei Kogyo, NPK = 13-3-11%) 5 g / 10 liters, borax 0.15 g / 10 liters of 10 cm in diameter filled with culture medium The seedlings were planted in pots. 2 per share
A strawberry seedling with two petioles and six leaves was used in the experiment.
The pathogen was infected by spraying conidia onto the aboveground parts of the plant. The experimental method is described in detail below. That is, the polysaccharide M-100-1 obtained in the same manner as in Example 2 was adjusted to a concentration of 10 μg / ml, and 1 ml per one was sprayed on the above seedlings. Anthracnose virulent Choletotrichum fragalier M6013 (FERM P-12
625), soluble starch 15 g / liter, yeast extract 4 g / liter, K ₂ HPO ₄ 1 g / liter, MgSO ₄ · 7a
q. Inoculate 100 ml of medium having a composition of 0.5 g / liter (pH 6) into a 500 ml flask,
After culturing at 5 ° C. for 5 days with shaking, the hyphae were removed by filtration with gauze, and those containing 10 ⁶ cfu / ml of conidia were used. The spore fluid of the pathogenic bacterium prepared in this manner is used as a polysaccharide M
3 days after spraying -100-1, spray inoculate 1 ml per strawberry seedling and keep the humidity at 100% for 2 days.
The inoculated spores were germinated. Subsequent cultivation was performed in a vinyl greenhouse at a temperature range of 20 to 35 ° C. by watering every day so that the water content of the soil in the pot was 80% of the maximum water capacity. 14 days after the inoculation of the pathogenic bacteria, the onset status was examined. In investigating the disease onset, the disease index indicating the degree of progression of the symptom was set as shown in Table 5, and the disease index was measured for all leaves and petiole used in the experiment, and per leaf and per petiole Was calculated. From this average disease incidence index, (non-treatment area disease index-treatment area disease index) / non-treatment area disease index was calculated and used as the control value. As a result, as shown in Table 6, a highly significant difference was found between the disease index of the treated group of polysaccharide M-100-1 and the untreated group, and most of the seedlings in the untreated group were While the anthracnose disease causes death, the treated area was suppressed from progressing.

Table 5. Setting of disease index of strawberry anthrax disease Disease index Leaf petioles 1 Number of lesions 1-10 Number of small lesions 1-2 2 Number of lesions 11-20 Number of small lesions 3-5 3 Number of lesions 21-30 Small lesions Number 6 or more 4 Number of lesions 31 or more Medium-large lesion formation 5 Leaf wilting due to large lesion Petiole breakage or leaf wilting due to large lesion

Table 6. Suppression of pathogenesis by polysaccharide M-100-1 Investigation site Polysaccharide M-100-1 Disease index Control value (%) Leaf-free treatment 3.7 0 Treatment 1.5 59 ** Petiole-free treatment 3.2 0 Treatment 1.2 63 ** (1 ward 10 shares) **: Highly significant difference by cumulative chi-square test

[0026]

Industrial Applicability The polysaccharide M-100 substance group of the present invention is a novel substance, and by contacting this polysaccharide M-100 substance group with a plant, the plant is resistant to the pathogen before it is contacted with the pathogen. Even if the pathogen is infected, the antibacterial substance such as chitinase does not allow the pathogen to grow in the plant even if the pathogen is infected, and it does not lead to disease, increasing the resistance of the plant to the pathogen. It shows an elicitor-like activity effect.

[Brief description of drawings]

FIG. 1 is an IR absorption spectrum of the substance group M-100 of polysaccharide of the present invention.

FIG. 2 is a UV absorption spectrum of the polysaccharide M-100 substance group of the present invention.

FIG. 3 shows the content of the polysaccharide M-100 substance group in the fraction obtained by gel filtration with Bio-GelA-5m from the bacterial cell components of Choletotrichum fragalier M-6013.

FIG. 4 shows the content of the polysaccharide M-100 substance group in the fraction obtained by gel filtration with Bio-GelP-30 from the bacterial cell components of Choletotrichum fragalier M-6013.

FIG. 5 shows the effect of polysaccharide M-100-1 on the induction of chitinase activity in tomato seedlings.

FIG. 6 shows the effect of the polysaccharide M-100-2 on the induction of chitinase activity in tomato seedlings.

FIG. 7 shows the effect of the polysaccharide M-100-3 on the induction of chitinase activity in tomato seedlings.

FIG. 8: Polysaccharide M-100 on cultured cells of tomato
The effect of -1 is shown.

Claims (3)

[Claims] 1. A polysaccharide M-100 substance group characterized by having the following physicochemical properties. Molecular weight: 7500 to 13000 Measured by gel filtration high performance liquid chromatography using Asahipak GS-510 (mobile phase: water, molecular weight standard: pullulan (Showa Denko)). Action: It has at least chitinase activity inducing activity. Constituent sugar: Glucose is hydrolyzed under conditions of 100% 1.2N HCl at 80 ° C for 24 hours, and free glucose is measured by high performance liquid chromatography using TOSOH Amide-80 (mobile phase: acetonitrile-water 60:40). Determined by Color reaction Phenol-sulfuric acid reaction: Positive Lowry reaction: Negative Elson-Morgan reaction: Negative Carbazole sulfuric acid reaction: Negative Solubility Water: Soluble, ethanol, acetone, chloroform, benzene: Insoluble Acid, neutral, basic Distinction: Medium Color of organic substance: White IR absorption spectrum: Figure 1. (KBr) 3400, 2950, 1670,
Absorption at each wave number near 1560, 1400 and 1350 cm ^-1 . UV absorption spectrum: Figure 2. No particularly characteristic peak (water: 1 mg / ml) 2. A polysaccharide M-100 substance group-producing bacterium belonging to the genus Choletotrichum and having the following physicochemical properties is cultured in a medium, and then the polysaccharide M-100 substance group is collected from the culture. A method for producing the polysaccharide M-100 substance group. Molecular weight: 7500 to 13000 Measured by gel filtration high performance liquid chromatography using Asahipak GS-510 (mobile phase: water, molecular weight standard: pullulan (Showa Denko)). Action: It has at least chitinase activity inducing activity. Constituent sugar: Glucose is hydrolyzed under conditions of 100% 1.2N HCl at 80 ° C for 24 hours, and free glucose is measured by high performance liquid chromatography using TOSOH Amide-80 (mobile phase: acetonitrile-water 60:40). Determined by Color reaction Phenol-sulfuric acid reaction: Positive Lowry reaction: Negative Elson-Morgan reaction: Negative Carbazole sulfuric acid reaction: Negative Solubility Water: Soluble, ethanol, acetone, chloroform, benzene: Insoluble Acid, neutral, basic Distinction: Medium Color of organic substance: White IR absorption spectrum: Figure 1. (KBr) 3400, 2950, 1670,
Absorption at each wave number near 1560, 1400 and 1350 cm ^-1 . UV absorption spectrum: Figure 2. No particularly characteristic peak (water: 1 mg / ml) 3. A polysaccharide M-1 belonging to the genus Choletotrichum
Bacteria produced by substance group 00 are Colletotrichum fragarie M
6013 (FERM P-12625).
The manufacturing method described.