JPH0124121B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a control agent for pear black spot. The agent for controlling pear black spot of the present invention is used in the cultivation of pears to prevent the occurrence of pear black spot and to recover pears suffering from pear black spot. [Technical Background and Description of Prior Art] Pear black spot is a plant disease that frequently occurs on pears, especially 20th century pears.
It causes black lesions on leaves and branches, and if it occurs on fruit, it softens, splits, and drops. For this reason, there is a need to control the occurrence of pear black spot on pear trees. Chitosan is a polysaccharide obtained by deacetylating chitin contained in the shells of crustaceans such as shrimp and crabs, and is a polysaccharide in which D-glucosamine is linked in a linear chain through β-1,4 bonds. Chitosan with a low degree of polymerization obtained by decomposing chitosan is also known. Methods for decomposing chitosan include chemical methods such as hydrolysis using hydrochloric acid, oxidative decomposition using nitrous acid, and oxidative decomposition using chlorine, and methods using enzymes (chitosanase). As a microorganism that produces chitosanase, Bacillus sp. R-4 [Tominaga et al.: Y. Tominaga et al.
et al: Biochimica et Biophysica Acta) No. 410
Vol. 145-155 (1957)], Penicillium islandicum (DMFenton et al.: Journal of General Microbiology)
al Journal of General Microbiology), Vol. 126, pp. 151-165 (1981)], Bacillus
sp.) 99-5 (Horiuchi: Japanese Society of Agricultural Chemistry, Abstracts of the 1981 Conference, p. 550), Streptomyces No. 6 [G.S. Price et al.: Journal of Bacteriology ( J.S.
Price al: Journal of Bacteriology) Volume 124, No.
1574-1584 (1975)], Streptomyces griseus [Otakara et al.: Chitin, Chitosan and Related.
Enzymes (A. Ohtakara et al: Chitin,
Chitosan and Related Enzymes) pp. 147-160 (1985) Academic Press] and Bacillus sp. No. 7-M (Patent Application 1985-120673)
Chitosan affects the growth of phytopathogenic fungi [P. Stoessel et al.: Phytopathologische Lift (P. Stoessel et al.
Zeitschrift, Vol. 111, pp. 82-89 (1984), CRAllan et al.
Mycology, Vol. 3, pp. 285-287 (1979)] and that decomposition products of chitosan affect the inhibition of mold growth on peas [D.F. Kendra et al.: Experimental Mycology (DF
Kendra et al Experimental Mycology) No. 8
Vol. 276-281 (1984)], and it has been proposed that chitosan and mildly decomposed products of chitosan inhibit bacterial growth (Japanese Patent Application No. 223749/1984); There are no detailed reports on the control of plant diseases using the decomposition products of . The present inventors have been conducting research on chitosan for many years, and in their research, they discovered that low-molecular-weight chitosan with a molecular weight of 3000 or less is effective in controlling the occurrence of pear black spot on pear trees. ,
The present invention was achieved based on this knowledge. [Object of the Invention and Summary of the Invention] An object of the present invention is to propose a drug effective for controlling pear black spot. The present invention is a control agent for pear black spot containing low-molecular chitosan with a molecular weight of 3000 or less as an active ingredient, and as the low-molecular chitosan, chitosan oligosaccharides with a degree of polymerization of 2 to 8 can be used, Alternatively, chitosan can be decomposed with chitosanase derived from Bacillus No. 7-M (Feikoken Bacteria No. 8139), and the obtained product can be used. [Detailed Description of the Invention] In theory, the low-molecular-weight chitosan having a molecular weight of 3000 or less, which is the active ingredient of the agent for controlling black pear blight of the present invention, can be prepared by subjecting chitosan to known methods such as acid decomposition, oxidative decomposition, or enzymatic decomposition. It can be obtained by decomposing it by
In the decomposition reaction, it is necessary to adjust the molecular weight of the low-molecular-weight chitosan product to 3000 or less. The molecular weight of low-molecular-weight chitosan can be adjusted by separating a fraction with a molecular weight of 3000 or less using a conventional method from a decomposed product of chitosan using a known method. It is also possible to obtain a product rich in low-molecular-weight chitosan with a desired molecular weight of 3000 or less by determining the conditions necessary to obtain the desired molecular weight and decomposing chitosan under the decomposition conditions. Chitosan oligosaccharide having a degree of polymerization of 2 to 8 can be obtained by the same method as the above-mentioned low molecular weight chitosan having a molecular weight of 3000 or less. The enzyme used in enzymatic decomposition to obtain low-molecular chitosan with a molecular weight of 3000 or less, and especially chitosan oligosaccharide with a degree of polymerization of 2 to 8, may be any chitosanase that can decompose chitosan into a low-molecular-weight chitosan. For example, any substance can be used, but it is convenient and economical to decompose it with chitosanase produced by Bacillus No. 7-M. Bacillus No. 7-M is Bacillus sp. No. 7, which was isolated from the soil of a virgin swamp in Unzen, Obama-cho, Minamitakagi-gun, Nagasaki Prefecture, as a bacterium that can grow in a medium containing chitin or chitosan as the sole carbon source. This parent strain was treated with N-methyl-N'-nitroso-N-nitrosoguanidine (NTG) to induce mutations, and among the streptomycin-resistant mutants obtained, highly active chitosanase was selected. This is a mutant strain that was isolated as one capable of producing , and has been deposited with the National Institute of Microbial Technology, Ministry of International Trade and Industry as FERM P-8139. The mycological properties of Bacillus No. 7-M are shown below. A Cell morphology (1) Cell shape and size: Short bacillus, (broth and broth agar slant culture, 37℃, 24~
(72 hour culture) (2) Presence or absence of cell pleomorphism: None, (3) Presence or absence of motility: Yes (Meat juice agar semi-fluid high-layer puncture culture) (4) Presence or absence of spores: Yes, endospores and Naked spores, spherical, [Dorner's staining method and modified Witz method] (5) Gram staining: positive, [broth agar slant culture, 37°C, 18 hours, modified Hucker method] B. Growth status in each medium (1) Broth agar plate culture (37°C, 24-168 hours):
It forms round, raised, milky-white colonies with a thread-like periphery. The surface of the colony is uneven, slightly shiny, and translucent. It will get better as time passes. Does not produce pigment. (2) Broth agar slant culture (37℃, 24-168 hours):
Forms milky-white colonies that are spread out. Colonies have convex circular ridges and are shiny. It grows well and will spread over time. Does not produce pigment. (3) Broth liquid culture (37℃, 24-168 hours): No film is formed on the surface. The whole thing becomes cloudy over time. Granular sediments are formed at the bottom and gradually increase in number. (4) Meat juice gelatin puncture culture (25°C, 24-168 hours): Grows along the puncture line and liquefies. The surface and interior grow funnel-shaped and liquefy. The liquefied part becomes cloudy. (5) Litmus milk (37℃, 24-168 hours): 2
After a few days, the upper part gradually liquefied, and on the fourth day, the color completely changed and became acidic. It does not coagulate. As time passed, liquefaction progressed and it became translucent. C Physiological properties (1) Nitrate reduction: - (Nitrate broth medium, 37℃, 24-120 hours) (2) Denitrification reaction: - (Komagata et al. method, using fermentation tube, 37℃, 24-120 hours)
(120 hours) (3) MR test: + (37℃, 24-168 hours) (4) VP test (acetylmethylcarbinol production test: + (37℃, 24-168 hours) (5) Indole production: - (37℃, 24-168 hours) (6) Hydrogen sulfide generation: - (TSI agar method, 37℃, 24-168 hours) (7) Starch hydrolysis: + (37℃, 24-168 hours) (8) Utilization of citric acid (Koser's medium, 37℃, 24-168 hours): - (Christensen's medium, 37℃, 24-168 hours): + (9) Utilization of inorganic nitrogen source (37℃, 24 hours) ~168 hours) Nitrate: undetermined, ammonium salt: undetermined, (10) Pigment production (mannitrate/yeast extract agar slant):
− [King A agar slant medium]: − (11) Presence or absence of fluorescence: None (12) Urease: + (Christensen-Urea agar medium, 37°C,
(24-168 hours) (13) Oxidase: + (broth agar, 37℃, 24-48 hours) (14) Catalase: + (broth agar, 37℃, 24-48 hours) (15) Growth range: (gravy agar medium) Temperature: undetermined, PH: 5-10, added salt concentration: undetermined, (16) Attitude towards oxygen: aerobic (1% glucose gravy high-rise agar medium, 37℃,
24-72 hours) (17) O-F test (Hugh-Leifson method, 37°C, P-glucose): Acid is produced fermentatively.
(fermentative) (18) Presence or absence of acid and gas generation from sugars (37℃, 24-168 hours):

【table】

[Table] The above mycological properties are described in Bergey's Manual of Determinative Bacteriology.
When searching the 8th edition (1974) of Bacteriology, it was found that strain No. 7-M most likely belongs to the genus Bacillus. The enzymatic chemical properties of chitosanase produced by Bacillus No. 7-M are as shown below. (1) Action: Acts on chitosan, arbitrarily decomposes β-1,4 bonds from the internal chains of the molecule, and mainly forms chitosan oligosaccharides (GlcN ₎ (n=2 to 8) (dimers to 8
(quantity). Chitosan oligosaccharides can be separated from the chitosan decomposition solution using high performance liquid chromatography. The decomposition degree of chitosan in this decomposition solution is about 45%. It also acts on carboxymethyl cellulose (CMC), degrading it to some extent, but has no effect on chitin. (2) Action temperature range and optimal action temperature: When soluble chitosan is used as a substrate, it works up to 80°C, and the optimal action temperature is 50°C. Figure 1 shows the relationship between temperature and specific activity when reacting for 10 minutes at pH 6.0. (3) Action PH range and optimum PH: It works in the range of PH3 to 9, and the optimum PH is PH
It is 6. 1 ml of 1% soluble chitosan and 2 ml of each pH buffer
FIG. 2 shows the relationship between pH and specific activity of the enzyme when a reaction solution containing 1 ml of the enzyme solution was reacted at 37° C. for 10 minutes. (4) Thermostability: Almost stable until kept at 50°C for 15 minutes; about 40% of the enzyme is inactivated by heating at 60°C for 15 minutes, and completely deactivated by heating at 70°C for 15 minutes. Deactivated. Figure 3 shows the relationship between temperature and specific activity. (5) PH stability: The remaining enzyme activity was measured after being left in a 0.1M buffer at 30°C for 2 hours.
It was stable within the range of 11. One of the major characteristics of chitosanase produced by Bacillus No. 7-M is that it is stable at pH 10 to 11. Figure 4 shows the relationship between PH and specific activity. (6) Inhibitor: Chitosanase produced by Bacillus No. 7-M was treated with HgCl ₂ , PbCl ₂ , at a final concentration of 1×10 ⁻³ M;
AgNO ₃ , and nearly 100 due to the presence of PCMB
% was inhibited. (7) Substrate specificity: Various substrates were used, and the final concentration of the substrate was 0.25%.
1 oxygen protein per 4 ml of enzyme reaction solution
The amount of total reducing sugars and hexosamine released after 1 hour (mg/mg protein/hour) was measured. The results are shown in Table 1.

[Table] Chitosanase produced by Bacillus No. 7-M degraded chloylda chitosan, soluble chitosan, and glycol chitosan well, and also slightly degraded carboxymethylcellulose (CMC), but had no effect on powdered chitosan. .
In addition, colloidal chitin, glycol chitin,
Powdered chitin and methylcellulose did not decompose at all. (8) Molecular weight: Figure 5 shows the results of measuring the molecular weight by SDS-polyacrylamide electrophoresis. In Figure 5, (○) is the molecular weight of chitosanase produced by Bacillus No. 7-M, which is approximately
It is 41000. Figure 6 shows the results of measuring the molecular weight by gel filtration using Sephadex G-100.
In Figure 6, (○) is the molecular weight of chitosanase produced by Bacillus No. 7-M, which is approximately
It is 30000. (9) Enzyme titer measurement method: 1g of powdered chitosan (28 mesh) was added to 50ml of
After dissolving in 0.1M acetic acid aqueous solution and adjusting the pH to 6.0 with 0.1M sodium acetate aqueous solution, add 0.1M acetate buffer (PH: 6.0) to bring the total volume to 100ml.
Prepare a 1% soluble chitosan solution of the substrate. Add 1 ml of the enzyme solution pre-incubated in the same manner to 1 ml of the 1% soluble chitosan solution of the substrate that was pre-incubated at 37°C for 5 minutes.
Allow the enzyme reaction to occur for exactly 10 minutes at ℃. Thereafter, the reaction solution is boiled for 3 minutes to stop the enzyme reaction, and the amount of reducing sugar produced in the reaction solution is quantified. The amount of enzyme that releases reducing sugar equivalent to 1 μmol of glucosamine under these conditions is defined as 1 unit of chitosanase activity. The agent for controlling pear blight of the present invention can be used in the form of a solid or liquid carrier, or a mixture with other agricultural chemicals. For effective control of pear black spot, it is preferred to spray pear trees in the form of a solution of at least 0.05%. When used in powder form, it is preferably used in conjunction with a spreading agent. The present invention will be explained in more detail below using reference examples and test examples that can be substituted for the examples. Reference Example 1 (Preparation of seed culture) Pour 50 ml of liquid medium (PH: 7.2) containing 0.8% yeast extract, 0.4% peptone, 0.2% meat extract, and 0.5% colloidal chitosan into a 250 ml Erlenmeyer flask.
After sterilization by a conventional method, Bacillus sp. No. 7-M (FFRM P-
8139) and cultured with shaking at 30°C for 1 day. (Preparation of culture solution for enzyme production) Pour one liquid medium with the same composition as above into two 5-volume Erlenmeyer flasks, sterilize it by a conventional method, and add 40 ml of the seed culture solution obtained above.
was inoculated and cultured with shaking at 30°C for 4 days. The culture solution was centrifuged at 6000 r.pm.
The bacterial cells were removed, and the chitosanase activity of the resulting supernatant was measured by the enzyme titer measurement method described above. The concentration was 0.99 units per ml of supernatant. (Purification of enzyme solution) The supernatant liquid obtained above was mixed, 1015 g of solid ammonium sulfate (corresponding to 80% saturation of ammonium sulfate) was added to the obtained mixed liquid 1.81, and the resulting precipitate was filtered. was dissolved in distilled water to make 177 ml. This enzyme solution was mixed with distilled water, followed by 0.02M phosphate buffer (PH: 6.0).
After dialyzing against
Column packed with CM-Sephadex C-50 equilibrated with 0.02M phosphate buffer [2.6 cm (diameter) x 45
cm (length)] to adsorb chitosanase.
Most of the impure proteins were concentrated in the pass-through section. After washing this column with 350 ml of 0.02M phosphate buffer, the enzyme protein was eluted with a linear concentration gradient of 0 to 0.5M sodium chloride. Next, the 218th to 240th fractions that showed chitosanase activity were combined and concentrated 17 times using an ultrafiltration device using a diaflow membrane filter PM-10 (manufactured by Amicon).
Gel filtration was performed using Sephadex G-100. The 50th gel filtration showed chitosanase activity.
63 fractions were combined and again subjected to column chromatography using CM-Sephadex C-50. Adsorb the enzyme under the same conditions as last time, and
The enzyme protein was eluted using a linear concentration gradient with 0.5M sodium chloride. Reference Example 2 (Preparation of chitosan oligosaccharide) Take 15 g of chitosan (degree of deacetylation: 99%) in a 500 ml beaker, and add 150 g of deionized water to it.
ml and 50ml of 1N lactic acid, and after stirring thoroughly,
Deionized water was added to bring the total volume to 300 ml. Take 10ml of this chitosan lactic acid solution into a test tube and
Pre-incubation was performed for 10 minutes in a constant temperature bath at ℃. Separately, the chitosanase solution obtained by column chromatography using CM-Sephadex C-50 in the reference example was diluted with water to 10.5 units/ml, 1 ml of the solution was placed in a test tube, and pre-incubated in the same manner as above. This was added to the above chitosan lactic acid solution and reacted in a constant temperature bath at 37°C.
After 100 minutes, the beaker was placed in a boiling bath for 6 minutes to stop the reaction, and the reaction solution was centrifuged, filtered, and freeze-dried to obtain 19.1 g of chitosan oligosaccharide powder. The polymerization degree of D-glucosamine in this chitosan oligosaccharide was 2 to 8, and D-glucosamine and chitosan with a polymerization degree of 9 or more were hardly contained. Test example (test for pear black spot) Pear black spot fungus [Alternaria alternata Japanese pear
The effect of low-molecular-weight chitosan on the formation of lesions was tested when bacterial cells of A. pathotype) were sprayed. (1) Preparation of sample (1-1) Preparation of low-molecular-weight chitosan solution Take 15 g of chitosan (degree of deacetylation: 95%) in a 500 ml beaker, add 150 ml of deionized water and 69 ml of 1M lactic acid, and stir thoroughly. After stirring and standing overnight, more deionized water was added to bring the total to 300 ml. This was pre-incubated at 37°C. Separately, the enzyme activity of the chitosanase solution obtained in Reference Example 1 was adjusted to 10.5 units/ml,
Pre-incubate 30 ml at 37°C and add this chitosanase solution to the previously prepared and pre-incubated chitosan solution.
After stirring at 37% for 50 minutes to react, the mixture was heated to boiling to stop the reaction. The reaction solution was centrifuged, further filtered, and then freeze-vacuum dried to obtain low-molecular-weight chitosan 20.8
I got g. Take 1 g of this low-molecular-weight chitosan, dissolve it in 100 ml of 0.5% acetic acid aqueous solution, and
-25 gel filtration using dextran as standard to fractionate low-molecular chitosan with a molecular weight of 3000 or less, neutralize it with an aqueous sodium hydroxide solution, pour it into ethanol to precipitate it, filter it, ethanol and Wash thoroughly with ether and dry overnight to obtain a powder of low-molecular chitosan with a molecular weight of 3000 or less.
I got g. Low molecular weight chitosan powder obtained here 0.1
g was taken and dissolved in 100 ml of water to obtain 100 ml of a 0.1% aqueous solution of low molecular weight chitosan. (1-2) Preparation of chitosan oligosaccharide solution 0.1 g of chitosan oligosaccharide powder obtained in Reference Example 2 (degree of polymerization: 2 to 8, average degree of polymerization: 5.5)
was dissolved in 100 ml of water to prepare 100 ml of a 0.1% aqueous solution of chitosan oligosaccharide. (1-3) Preparation of CM-chitosan solution 15 g of chitin powder was dissolved in 300 ml of 42% sodium hydroxide aqueous solution, left to stand under reduced pressure at room temperature for 5 hours, stirred overnight at normal pressure, and filtered with a graph filter. did. Take the filter cake in a 1 volume beaker and add water to it.
After 200 g was added and left to stand for 30 minutes, the proportion of sodium hydroxide was adjusted to 14% and thoroughly stirred. While cooling the beaker in ice water, an aqueous monochloroacetic acid solution obtained by adding 23 g of monochloroacetic acid to 23 ml of water was gradually added dropwise thereto, and the mixture was left to stand for 30 minutes, and then left to stand at room temperature overnight. Neutralize this with acetic acid and
After dialysis for days, it was concentrated under reduced pressure, and the concentrated solution was freeze-dried in vacuum to obtain 14.7 g of CM-chitin (carboxymethyl-chitin). 500ml of 2.5g of CM-chitin obtained here
Add 300 ml of 14% sodium hydroxide aqueous solution to dissolve, stir at 85°C for 20 hours using an oil bath,
Made it react. After the reaction, the mixture was neutralized with acetic acid, dialyzed for 3 days, concentrated, and the concentrated solution was dried to obtain 1.9 g of CM-chitosan. Dissolve this CM-chitosan in 100ml of water,
100 ml of 0.1% aqueous solution of CM chitosan was prepared. (2) Test method Bat [30 (horizontal) x 25 (vertical) x 4 (depth) cm]
A 3.5 cm thick sponge of the same size was placed in the container, tap water was poured into it until the surface of the sponge was wetted, and young leaves of a sensitive variety of Nijisseiki pear were placed on top of the sponge. Pear black spot fungus [Alternaria alternaita]
Japanese Peer Pasotype (Alternaria)
alternata Japanese pear pathotype] were suspended in the above sample solution at a spore concentration of 10 ⁶ /ml, and this spore suspension was sprayed onto young leaves of Nijisseiki pear arranged on a sponge in a vat. After incubating at 26°C for 24 hours, the lesions of pear black spot formed on the young leaves of Nijisseiki pears were counted on three leaves in one plot. As a control group, without adding sample solution,
A spore suspension obtained by suspending the conidia of the Pear Spot fungus in water at the same spore concentration as above,
After spraying the young leaves of Nijisseiki pear in the same way as above,
This was incubated in the same manner as the test plot described above, and the lesions of pear black spot formed on the young leaves of Nijisseiki pears were counted. The inhibition rate of lesion formation was calculated using the following formula. Suppression rate (%) = (1 - number of lesions per unit area in treated area/number of lesions per unit area in control area) x 100 (3) Test results The test results were as shown in Table 1. .

〔Effect of the invention〕

It is possible to control the outbreak of pear black spot, which is a concern in cultivating Nijisseiki pears.

[Brief explanation of drawings]

Figure 1 is a chart showing the relationship between temperature and specific activity in the action temperature range of chitosanase produced by Bacillus No. 7-M, and Figure 2 is a chart showing the relationship between PH and specific activity in the action PH range of chitosanase. Figure 3 is a diagram showing the relationship between temperature and specific activity in the thermostability of chitosanase, and Figure 4 is a diagram showing the relationship between PH and specific activity in the PH stability of chitosanase. Figure 5 is a chart showing the results of molecular weight measurement of chitosanase by SDS-polyacrylamide electrophoresis, and Figure 6 shows
The figure is a chart showing the results of the molecular weight measurement of chitosanase by the gel filtration method.

Claims (1)

[Scope of Claims] 1. A control agent for pear black spot, characterized in that the active ingredient is low-molecular chitosan with a molecular weight of 3000 or less. 2. The agent for controlling pear black spot according to claim 1, wherein the low-molecular-weight chitosan is a chitosan oligosaccharide in which the degree of polymerization of D-glucosamine is 2 to 8. 3 Low-molecular-weight chitosan transforms chitosan into Bacillus No.7
Pear black spot according to claim 1 or 2, which is obtained by decomposing with chitosanase derived from -M (Feikoken Bacteria No. 8139). repellent.