JP6997456B2 - Compositions for enhancing plant disease resistance or for controlling plant diseases and their usage methods - Google Patents

Description

The present invention provides a method for enhancing plant disease resistance or controlling plant disease by using esterase and / or xylanase.

Plants are equipped with a biological defense system that is similar to the immunity of animals, and when this function is activated, the plant activates various defense systems and becomes a plant that is less susceptible to disease. Recently, in the development of fungicides, from the viewpoint of environmental protection and avoidance of drug-resistant bacteria, there is an active search for drugs that try to control diseases by utilizing their defense systems. These drugs have (1) a preventive effect against multiple pathogens (wide spectrum of action), (2) an extremely low incidence of resistant bacteria, and (3) a long-lasting control effect. , (4) It has the characteristic that it has little direct impact on the ecosystem itself. On the other hand, since it is difficult to search for and evaluate compounds having such activity as compared with bactericidal pesticides, the number of compounds that have been put into practical use is limited. In addition, all the drugs marketed in Japan are targeted at rice, but similar drugs are required for various crops.

In addition, the surface layer of plants is covered with a cuticle layer and wax to protect themselves from external stress, especially osmotic stress caused by transpiration and rainfall, invasion of pathogens, and feeding damage by pests. The enzyme that hydrolyzes cutin (fatty acid polyester) contained in this cuticle layer is called cutinase.

Recently, techniques for rapidly decomposing biodegradable plastic agricultural materials after use by enzymes obtained from plant-resident yeast and filamentous fungi have been developed (Patent Documents 1 and 2). In addition, techniques for mass-producing a plurality of enzymes have also been developed (Patent Documents 3 and 4, Non-Patent Document 1), and it is expected that inexpensive enzyme solutions will be available in large quantities on the market. Then, when a culture filtrate containing a high concentration of these biodegradable plastic-degrading enzymes derived from filamentous fungi and yeast is treated on a plant, a phenomenon has been found in which the plant dies. At this time, the cuticle layer is thin and easily infected with phytopathogenic bacteria. Therefore, an enzyme that can decompose a part of the cuticle layer, such as a biodegradable plastic-degrading enzyme, is used as a herbicide for exterminating harmful plants. It can be used (Patent Document 5). It has also been found that a culture medium containing a biodegradable plastic-degrading enzyme derived from yeast Pseudozyma antarctica also contains xylanase (hemicellulose-degrading enzyme) (Non-Patent Document 2). However, no yeast-derived biodegradable plastic-degrading enzyme that actually has cutin-degrading activity has been confirmed. In addition, a method for enhancing plant disease resistance and controlling plant disease with a biodegradable plastic-degrading enzyme has not been known.

Patent No. 4915593 Patent No. 5082125 Patent No. 58492297 International Publication No. 2014/109360 Japanese Unexamined Patent Publication No. 2014-129287

Journal of Oleo Science (2016), 65 (3) 257-262 AMB Express (2015), 5:36

Until now, the range of applications of biodegradable plastic-degrading enzymes and cultures of microorganisms such as filamentous fungi or yeasts containing them has been limited, and the potential of the enzymes and cultures has not been fully utilized. It is an object of the present invention to provide a novel use of a biodegradable plastic degrading enzyme and a culture of a microorganism containing the same.

As a result of diligent studies to solve the above problems, the present inventors surprisingly found that a culture of microorganisms used as a biodegradable plastic-degrading enzyme was contained in an esterase and / or The present invention has been completed by finding that it exerts an infection-suppressing action and a pathogenic fungus germination-suppressing action on pathogens through the action of xylanase, and increases the expression of stress-resistant genes in plants.
That is, the present invention provides the following composition for enhancing plant disease resistance or plant disease control, and a method for enhancing plant disease resistance or controlling plant disease using them. be.
[1] A composition for enhancing plant disease resistance or controlling plant diseases, which comprises esterase and / or xylanase.
[2] The composition according to the above [1], wherein the esterase contains a biodegradable plastic degrading enzyme.
[3] The composition according to the above [2], wherein the biodegradable plastic degrading enzyme is selected from the group consisting of a cutinase and a cutinase-like enzyme.
[4] The composition according to any one of the above [1] to [3], wherein the esterase and / or the xylanase is a microbial culture solution or an extract.
[5] The microorganisms include the genus Pseudozyma, the genus Paraphoma, the genus Cryptococcus, the genus Mucor, the genus Humicola, the genus Thermomyces, the genus Thermomyces, and the genus Tala. The genus Chaetomium, the genus Torula, the genus Sporotichum, the genus Malbranchea, the genus Alternaria, the genus Cladosporium, the genus Penicillium. ), The composition according to the above [4], which is selected from the group consisting of the genus Pseudomonas, the genus Bacteroides, and the genus Acidovorax.
[6] The composition according to any one of the above [1] to [5], wherein the esterase concentration is 0.005 to 0.5 U / mL.
[7] The composition according to any one of the above [1] to [6], which comprises both the esterase and the xylanase.
[8] The composition according to any one of the above [1] to [7], wherein the plant has a cuticle layer.
[9] The composition according to any one of the above [1] to [8], wherein the plant is a higher plant.
[10] A method for enhancing plant disease resistance or controlling plant disease, which comprises a step of treating a plant with the composition according to any one of the above [1] to [9].
[11] The method according to the above [10], wherein the treatment step comprises a step of spraying the composition onto the plant.
[12] The method according to [11] above, wherein the spraying step comprises spraying 0.1 to 1 mL of the composition per leaf of the plant.

According to the present invention, esterase, which is a biodegradable plastic-degrading enzyme contained in a culture of microorganisms, and / or xylanase contained in the culture enhances disease resistance of plants or enhances disease resistance of plants. It can control plant diseases. Although plant disease resistance enhancers have been studied for a long time, only three kinds of drugs targeting rice are currently known as products in Japan. However, the disease resistance enhancing action or disease controlling action by esterase and / or xylanase of the present invention can be used not only in grasses but also in a wide range of crop species such as tomato, white sardine, and tobacco, and is versatile. high. Further, since esterase and xylanase, which are the active ingredients of the present invention, are proteins rather than small molecule compounds known as conventional disease resistance enhancers, they are easily decomposed in nature and are small molecules. There is no persistence in the environment caused by compounds, etc., and the environmental load is low.

A photograph showing changes in leaf symptom. A graph showing changes in leaf symptom. Micrograph of pathogen spores. Graph of germination rate of pathogen spores. Graph of the amount of DNA derived from a pathogen. A photomicrograph of a leaf containing a pathogen. Graph of plant stress resistance genes. Graph of active oxygen species response genes of plants. A photo of the leaves. Graph of the degree of morbidity. A photo of the leaves. Graph of the degree of morbidity. A photo of the leaves. Graph of lesion diameter. A photo of the leaves. Graph of the degree of morbidity. A photo of the leaves. Graph of the degree of morbidity.

The present invention relates to a composition for enhancing plant disease resistance or controlling plant diseases, which comprises esterase and / or xylanase.
As used herein, the term "esterase" refers to a hydrolase that decomposes an ester into an acid and an alcohol by a chemical reaction with water. As the esterase of the present invention, various esterases can be used without limitation as long as they have an activity of decomposing a part of the cuticle layer, and are used, for example, as a biodegradable plastic-degrading enzyme. You may adopt the enzyme that is present.

As used herein, the term "biodegradable plastic-degrading enzyme" refers to an enzyme having an activity of degrading biodegradable plastic. In the composition of the present invention, various enzymes can be used without limitation as long as they have an activity of degrading biodegradable plastics, and for example, cutinase or cutinase-like enzyme may be adopted. The activity of degrading the biodegradable plastic can be determined by measuring the degrading activity of the polybutylene succinate-co-adipate (PBSA) emulsion, or by measuring PBSA, polybutylene succinate, polybutylene adipate terephthalate, polylactic acid and the like. It can be determined by measuring the decomposition activity of the biodegradable plastic film of.

As used herein, the term "xylanase" refers to an enzyme having an activity of decomposing xylose into xylose, and can decompose hemicellulose in a plant cell wall. In the composition of the present invention, various xylanases can be used without limitation as long as they are enzymes having an activity of decomposing hemicellulose.

The esterase and the xylanase can be produced by microorganisms. In the composition of the present invention, esterase and / or xylanase isolated and purified from the microorganism may be used, and the culture solution (culture filtrate) or extract of the microorganism is used as esterase and / or xylanase. May be good. The microorganism that produces the esterase and / or the xylanase is not particularly limited, and is, for example, the genus Pseudozyma, the genus Paraphoma, the genus Cryptococcus, the genus Mucor, and Fumicola. (Humicola), Thermomyces, Talaromyces, Chaetomium, Torula, Sporotichum, Malbranke (Malbran) Consists of the genus Cladosporium, the genus Penicillium, the genus Pecilomyces, the genus Pseudomonas, the genus Bacteroides, and the genus Acidovorax. There may be.

The biodegradable plastic-degrading enzyme as the esterase is preferably Pseudozyma antagonist (for example, GB-4 (1) W strain, GB-4 (0) -HPM7 strain (Patent Microorganism Deposit, National Institute of Technology and Evaluation). Yeast deposited at the center; deposit number NITE BP-02238) and OMM62-2 strain (yeast deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary; accession number NITE BP-02239), etc.) Estelase (PaE), Paraphoma genus related bacterium cutinase-like enzyme (PCLE), or cutinase-like enzyme 1 (CmCut1). PCLE is a Paraphoma strain such as Paraphoma-related strain B47-9 (filamentous fungus deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary; Accession No. NITE P-573; see Patent No. 5082125 if necessary). CmCut1 is an enzyme produced by genus related bacteria, and CmCut1 is a Cryptococcus magnus such as Cryptococcus magnus strain BPD1A (yeast deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary; Accession No. NITE P-02134). Or an enzyme produced by its relatives.

The concentration of the esterase in the composition of the present invention can be appropriately adjusted according to the type of the plant to be applied and the type of the pathogen thereof. The concentration of the esterase may be, for example, 0.005 to 0.5 U / mL, preferably 0.006 to 0.2 U / mL, and more preferably 0.007 to 0.07 U / mL.
The titer of esterase shown here is measured by measuring the amount of decrease in turbidity of polybutylene succinate-co-adipate (PBSA) emulsion (Showa Denko KK, EM-301), which is a biodegradable plastic. It has been decided. The titer when the value of OD660 nm is decreased by 1 per minute is defined as 1U. As the buffer solution for measuring the enzyme activity, for example, Tris-hydrochloric acid buffer solution (20 mM Tris-HCl, pH 6.8, with or without calcium chloride (2 mM)) may be used.

The concentration of the xylanase in the composition of the present invention can be appropriately adjusted according to the type of the plant to be applied and the type of the pathogen thereof. The concentration of the xylanase may be, for example, 0.001 to 5.0 U / mL, preferably 0.005 to 1.0 U / mL.
The titer of xylanase shown here is the amount of xylose produced when xylan is decomposed. Biol. Chem. It was measured by an improved method of the Somogi-Nelson method as shown in (1980), 44 (12), 2943-2949. The titer to produce 1 μmol of xylose per minute was defined as 1 U.

In the composition of the present invention, the esterase or the xylanase may be used alone, or both may be used in combination. By using both enzymes having different mechanisms of action in combination, a synergistic plant disease resistance enhancing action and / or a plant disease controlling action is exhibited. When the esterase and the xylanase are used in combination, the isolated and purified esterase and the xylanase may be blended in the same composition, respectively, but a microbial culture solution or an extract containing these enzymes from the beginning is used. Then it is convenient. Alternatively, the composition containing only the esterase and the composition containing only the xylanase may be used simultaneously or continuously.

As used herein, the term "enhanced disease resistance of a plant" refers to inducing a situation in which infection is not established or symptoms are alleviated even if an arbitrary pathogen comes into contact with the plant. "Enhanced plant disease resistance" refers to, for example, elevation of injury response genes such as PIN2 (proteinase inhibitor 2) and LapA1 (leucine oxygen protected A1), elevation of PR4 (patogenesis reacted 4) and PRB1b, and disease response. In some cases, such as the release of reactive oxygen species, enhanced physiological response of plants to disease is an indicator. In addition, the term "plant disease control" described in the present specification means to suppress changes in symptom symptoms in plants as a result, regardless of the mechanism of action. The mechanism of action for controlling plant diseases includes, for example, changes in the physiological response of plants as described above, changes in the symbiotic microbial flora, or attenuation of pathogens, but is not limited thereto. do not have.

The composition of the present invention can be used for various plants because it has a non-specific effect of enhancing plant disease resistance and a plant disease control effect regardless of the type of plant or the type of disease. The composition of the present invention may be used for, for example, a plant having a cuticle layer, or a higher plant such as a seed plant (angiosperm or angiosperm) and a fern plant. In addition, the composition of the present invention comprises various agricultural products such as Aoi family, Akaza family, Abrana family, Ayame family, Isomatsu family, Rice family, Iwatobacco family, Ukogi family, Uri family, Kakinoki family, Kiku family, and Walnut family. , Kuwa family, Keshi family, Sesame family, Sakurasou family, Satoimo family, Cactus family, Perilla family, Shukaidou family, Ginger family, Water lily family, Violet family, Seri family, Senryo family, Tsutsuji family, Tsubaki family, Todaigusa family, Family, Nadesico family, Rose family, Higanbana family, Hirugao family, Fuurosou family, Vine family, Beech family, Button family, Matatabi family, Bean family, Mikan family, Yamanoimo family, Yukinoshita family, Yuri family, Orchid family, Ryuzetsuran family, It may also be used for agricultural products belonging to a family selected from the group consisting of the Lindou family. The composition of the present invention can enhance resistance to various diseases such as mold disease, bacterial disease, and viral disease, or control such various diseases.

The composition of the present invention may further contain any other component as long as it does not inhibit the disease resistance enhancing action or the plant disease controlling action of the plant. In addition, the composition of the present invention may further contain other active ingredients in order to further enhance the disease resistance enhancing action or the plant disease controlling action of the plant.

In some embodiments, the invention also comprises a method of enhancing plant disease resistance or controlling plant disease, comprising treating the plant with a plant disease resistance enhancing composition or a plant disease controlling composition. Related. As the method of the treatment, various methods can be used without limitation as long as it is a method for enhancing the disease resistance of the plant or controlling the disease of the plant as a result. For example, the treatment step may include spraying the composition onto the plant. The application site of the composition is not particularly limited, but may be, for example, the leaves, stems, fruits, or the like of the plant. The amount of the composition applied may be appropriately adjusted depending on the method of application and the site of application, and for example, 0.1 to 1 mL may be sprayed per leaf of the plant.

Hereinafter, the present invention will be specifically described with reference to Examples, but the scope of the present invention is not limited to these Examples.

<Example 1>
(1) Preparation of culture filtrate of Pseudozyma antarctica (P. antarctica) P. Antarctica (GB-4 (1) W strain) was precultured in YM medium (yeast extract malt extract medium) shown in Table 1.

To a 5 L volume of jar fermenter, 3 L of the medium having the composition shown in Table 2 was added, and the above P.I. Antarctica preculture solution was inoculated with 30 mL and cultured at 30 ° C., a stirring speed of 500 rpm, and an aeration rate of 8 LPM.

In order to prevent the outflow of the culture solution due to the formation of high foam, a 50-fold diluted solution of defoaming agent (trade name "Shin-Etsu Silicone KM-72F", manufactured by Shin-Etsu Chemical Co., Ltd.) is intermittently applied using a defoaming sensor. By 72 hours after the start of culturing, approximately 40 mL was added.
In addition, the sensor detects the decrease in pH due to the consumption of ammonium ions in the medium, and in order to add a nitrogen source and adjust the pH, ammonia water is automatically added dropwise to the medium as an alkali adjusting solution to adjust the pH to 6.0. did. From 24 hours after the start of culturing, the fed medium having the composition shown in Table 3 was fed at a rate of 0.5 L / d. Finally, the culture broth after culturing for 72 hours was filtered through a CELLULOSE ACETATE filter paper (manufactured by ADVANTEC) having a pore size of 0.45 μm to prepare a culture filtrate. This culture filtrate was used in each of the following tests. In addition, P. Even if another strain of Antarctica (GB-4 (0) -HPM7 strain or OMM62-2 strain) is used, a culture filtrate is prepared in the same manner as when the GB-4 (1) W strain is used. And could be used in the same way.

(2) Spray treatment on cut leaves P. Dilute antarctica culture filtrate (esterase titer: 8.0 U / mL) with 20 mM Tris-hydrogen buffer (pH 8.8) to 0.01 U / mL, 0.1 U / mL, 1.0 U / mL. A solution was prepared. 0.6 mL of each solution of 0.01 U / mL to 8.0 U / mL was sprayed on one leaf cut from dwarf tomato (MicroTom), and kept warm while moisturizing in a closed container (humidity). 100%, temperature 22 ° C.). The control group was sprayed with a culture filtrate in which the enzyme was inactivated by autoclave treatment. 72 hours (3 days) after spraying, spores (5 x 10 ⁴ spores / mL) of pathogenic filamentous fungi (Botrytis cinerea, B.c.) were applied to each cut leaf. Sprayed 0.6 mL each. Five cut leaves were used for each treatment group, and the appearance of the leaves was observed 10 days after the enzyme treatment, that is, 7 days after the spore treatment. Symptoms of brown lesions (sometimes covered with mold), yellowing of leaves with no obvious signs but fading green of leaves, no signs or yellowing The state of not being changed was evaluated as unchanged. FIG. 1A shows photographs of cut leaves subjected to spray treatment of pathogen spores after each enzyme treatment, and FIG. 1B shows the percentage of observed cut leaf states (no change, yellowing, or symptom). The graph is shown. As can be understood from these figures, in the 1.0 U / mL and 8.0 U / mL treatment groups, the infection efficiency was promoted and the symptom was promoted, but in the 0.1 U / mL treatment group, it was the same as the control group. It was a degree of symptom, and the symptom on the 10th day was alleviated in the 0.01 U / mL treatment group as compared with the control group.

(3) Germination rate of pathogens The mold spores and hyphae on the leaf surface on the second day after inoculation with the spores of the pathogen (B.c.) were stained with trypan blue. Four cut leaves were used for each treatment group and observed under a microscope. FIG. 2A shows an optical microscopic observation photograph of mold spores on the leaf surface 2 days after spraying the pathogenic bacterial spores after each enzyme treatment, and FIG. 2B shows a graph of the average value of the germination rate of the observed mold spores (error range is: The standard error calculated from 4 tests) is shown. Here, the different alphabets (a, b, and c) in FIG. 2B are significantly different in germination rate among the treatment groups as a result of performing a significance test at a significance level of 0.05 by Tukey's test. The same alphabet indicates that there is no significant difference in germination rate between the treatment groups. Specifically, in all of the 0.01 U / mL, 0.1 U / mL, and 1.0 U / mL treated groups, the germination of pathogenic spores was significantly suppressed as compared with the control group and the 8.0 U / mL treated group. (FIGS. 2A and 2B). In the control group and the 8.0 U / mL treatment group, pathogen spores were observed to germinate and the germination tubes spread on the leaf surface. Especially in the 8.0 U / mL treatment group, the germination rate was significantly higher than that in the control group.

(4) Abundance of pathogen in leaves DNA was extracted from the leaves on the third day after spore inoculation of the pathogen (B.c.), and the amount of DNA of the pathogen was quantified by qPCR. Five cut leaves were used for each treatment plot. FIG. 3 shows the analysis results using the β-tubulin gene of Gray mold disease as an index, that is, the average value of the relative values of the β-tubulin gene amount with respect to the correction by the Actin gene amount of tomato (the error range is calculated from 5 tests). The standard error) is shown. As a result, the amount of the pathogen is remarkably high in the 1.0 U / mL and 8.0 U / mL treatment groups, and it can be confirmed that the patient is infected with the pathogen. On the other hand, it was confirmed that the pathogens were clearly suppressed in the 0.01 U / mL and 0.1 U / mL treated groups as compared with the control group.

In addition, in order to investigate the presence of pathogens in the cross section of the leaves, four cut leaves were embedded in paraffin for each treatment group, and leaf sections (thickness 10 μm) were prepared and stained with thionin orange G. Mold spores and hyphae can be stained dark purple with thionin and plant cell walls can be stained orange with orange G. As a result of observing the prepared leaf sections with an optical microscope, it was observed that pathogens invaded the inside of the leaves in the 1.0 U / mL and 8.0 U / mL enzyme-treated groups, but 0.01 U / mL. In the 0.1 U / mL treatment group, the invasion of pathogenic bacteria was similar to that in the control group (Fig. 4).

(5) Plant response analysis RNA was extracted from the leaves on the third day after spore inoculation of the pathogen (B.c.), and various stress resistance-related genes (injury response genes PIN2 and LapA) of the plant were extracted by qRT-PCR. In addition, the expression levels of the disease response genes PR4 and PRB1b) were examined (corrected by the amount of Actin gene in tomato). Three cut leaves were used for each treatment plot. FIG. 5 shows the average value of the relative expression levels of various stress resistance-related genes with respect to the Actin gene of tomato (the error range is the standard error calculated from three tests). As a result, it was confirmed that the expression of the stress resistance-related gene was increased according to the culture filtrate treatment concentration.

It is known that plants release not only stress resistance-related genes but also reactive oxygen species response genes because they release active oxygen when they detect destruction of the cuticle layer or elicitor. When the change in the expression level of the reactive oxygen species response gene was examined by qRT-PCR using the extracted RNA, peroxidase (POD) and superoxide for the Actin gene of tomatoes were examined, especially by treatment with a culture filtrate of 0.01 U / mL. It was confirmed that the average value of the relative expression level of dismutase (SOD) (the error range is the standard error calculated from three tests) increased (Fig. 6).

(6) Degradation activity of cutin P. It has been confirmed that fatty acids having a carbon chain length of 16 or 18 are extracted from the cuticle layer when treated with an Antarctica culture filtrate (Ueda et al., Appl. Microbiol. Biotechnol., 2015). Therefore, regarding the cutin (fatty acid polyester) among the components constituting the cuticle layer on the surface of the plant, P.I. The degradability of Antarctica by the culture filtrate was examined. When cutin prepared from tomato peel was treated with the culture filtrate, ω-hydroxyhexadecanoic acid, which is a decomposition product of cutin, was detected in the reaction solution. From this, P. It was found that the culture filtrate of Antarctica has an activity to release the cutin monomer from the plant.

<Example 2>
(1) Effect on monocotyledonous plants and their pathogens P. The culture filtrate of Antarctica was diluted in the same manner as in Example 1. A solution of 0.01 U / mL with an esterase titer was sprayed on the leaves of the gramineous plant, Oat, at 0.6 mL, and kept warm while moisturizing in a closed container (humidity 100%, temperature 22 ° C). .. The control group was sprayed with a culture filtrate in which the enzyme was inactivated by autoclave treatment. Seventy-two hours after spraying, the cells were immersed in a culture solution (OD610: 0.65) of a pathogenic bacterium, streak blight, which infects the monocotyledonous plant Embaku. The same test was repeated twice using 4 cut leaves for each treatment plot. A state in which elliptical brown spots (spots) spread vertically was observed as a symptom. In addition, the lesion area was observed and the degree of disease was calculated based on the following formula (for the method of calculating the degree of disease, "rice, wheat, etc. fungicide field test method" (Japan Plant Protection Association, 2004) (Issued in March, 2014)).
Disease rate = 100 x (0 x n0 + 0.5 x n1 + 1 x n2 + 2 x n3 + 3 x n4 + 4 x n5) / 4N
n0: Number of leaves without lesions
n1: Number of leaves with lesions less than 1/8 of the leaves
n2: Number of leaves whose lesions are around 1/4 of the leaves
n3: Number of leaves whose lesions are around 1/2 of the leaves
n4: Number of leaves whose lesions are around 3/4 of the leaves
n5: Number of leaves with lesions of 7/8 or more of leaves
N: Total number of leaves investigated FIG. 7A shows a photograph on the third day after inoculation with pathogenic bacteria after each enzyme treatment, and FIG. 7B shows a photograph calculated from lesions observed on the third day after inoculation with pathogenic bacteria. The graph of the degree of morbidity is shown. As can be understood from these figures, in the 0.01 U / mL treatment group, the symptoms on the third day of inoculation were alleviated as compared with the control group, and the degree of onset was reduced.

(2) Effects on Brassicaceae plants and their pathogens P. The culture filtrate of Antarctica was diluted in the same manner as in Example 1. A solution of 0.01 U / mL with an esterase titer was sprayed on the rosette leaves of Arabidopsis thaliana, which is a brassicaceae plant, at 0.6 mL, and kept warm while moisturizing in a closed container (humidity 100%, temperature 22 ° C.). ). The control group was sprayed with a culture filtrate in which the enzyme was inactivated by autoclave treatment. 72 hours after spraying, spores (5 × 10 ⁴ spores / mL) of pathogenic filamentous fungi (Botrytis cinerea) were sprayed. The same test was repeated twice using 8 cut rosette leaves for each treatment plot. The condition in which brown lesions were formed was observed as a symptom, and the degree of morbidity was calculated based on the following formula. See Plant Protection Association, published in February 2003).
Disease rate = 100 x (0 x n0 + 1 x n1 + 2 x n2 + 3 x n3 + 4 x n4) / 4N
n0: Number of leaves without lesions
n1: Number of leaves with few (several) lesions
n2: Number of leaves with lesions less than 1/4 of the leaves
n3: Number of leaves with lesions less than 1/4 to 1/2 of the leaves
n4: Number of leaves whose lesions are more than half of the leaves
N: Total number of leaves investigated FIG. 8A shows a photograph on the third day after spraying the pathogenic spores after each enzyme treatment, and FIG. 8B shows the degree of disease onset calculated from the lesions observed on the third day after spraying the pathogenic spores. The graph of is shown. As can be understood from these figures, in the 0.01 U / mL treatment group, the symptoms on the 7th day after spore spraying were alleviated as compared with the control group, and the degree of onset was reduced.

(3) Effect on plant disease virus P. The culture filtrate of Antarctica was diluted in the same manner as in Example 1. A solution of 0.01 U / mL with an esterase titer was sprayed on the leaves of the Solanaceae plant, Samsun NN, at 0.6 mL, and kept warm while moisturizing in a closed container (humidity 100%,). Temperature 20 ° C). The control group was sprayed with a culture filtrate in which the enzyme was inactivated by autoclave treatment. 72 hours after spraying, a solution of the plant disease virus tobacco mosaic virus (TMV-OM strain) (4 μg / mL) was mechanically inoculated using carborundum. In order for the plant to suppress the spread of the virus, the lesions formed by the necrosis of the plant itself were observed, and the diameters of at least 57 lesions per leaf were measured to determine the average diameter. Three cut leaves were used for each treatment plot.
FIG. 9A shows a photograph showing the lesion on the 5th day after the virus inoculation, and FIG. 9B shows the average value of the average diameter (mm) of the lesion on each of the three leaves measured on the 5th day after the virus inoculation. The graph (the error range is the standard error calculated from three tests) is shown. Here, the asterisk in FIG. 9B indicates that the lesion was significantly smaller than that of the control group as a result of performing the significance test at the significance level of 0.01 by the t-test. That is, in the 0.01 U / mL treated group, the size of the lesion 5 days after inoculation was significantly smaller than that in the control group (FIGS. 9A and 9B).
From the above results, the composition for enhancing disease resistance or controlling plant diseases of the present invention has a non-specific effect of enhancing resistance to various diseases such as mold disease, bacterial disease, and viral disease. And it was found to have a control effect.

<Example 3>
P. In order to confirm what of the components contained in the culture filtrate of Antarctica contributes to the disease resistance enhancing action and the plant disease controlling action of plants, P.I. PaE, which is an esterase, and xylanase were separated and purified from the culture filtrate of Antarctica (see the preparation method described later). PaE was diluted to 0.01 U / mL with an esterase titer using 20 mM Tris-hydrochloric acid buffer (pH 8.8). Xylanase is described in P.I. Considering the test results using the culture filtrate of antarctica, 20 mM Tris hydrochloride buffer (pH 8.8) was used so as to have a concentration range considered to be effective for enhancing the disease resistance of plants or controlling plant diseases. , Diluted to 0.01 U / mL with a titer of xylanase. In addition, PCLE, which is a cutinase-like enzyme derived from the filamentous fungus Paraphoma genus B47-9 strain (accession number NITE P-573), is also prepared (see the preparation method described later). Prepared in mL. It is known that PCLE has a high substrate decomposing activity in the presence of calcium chloride when PBSA, which is a polyester, is used as a substrate. Therefore, the test was conducted with or without 2 mM calcium chloride in the solution. As a control group, the buffer solution (20 mM Tris-HCl, pH 8.8) used for dilution was used.

0.6 mL of each prepared enzyme solution was sprayed on the leaves of Gramineae plant, Enbaku, and kept warm while moisturizing in a closed container (humidity 100%, temperature 22 ° C.). 72 hours after spraying, the cells were immersed in a culture solution (OD610: 0.65) of streak blight that infects monocotyledonous plants. Six cut leaves were used for each treatment plot. FIG. 10A shows photographs of the 4th day after inoculation of pathogenic bacteria in various enzyme treatments, and FIG. 10B shows a graph of the degree of onset calculated from lesions observed on the 4th day after inoculation of pathogenic bacteria. As can be understood from these figures, the symptoms on the 4th day of inoculation were alleviated in the PaE-treated group, the xylanase-treated group, and the PCLE (with calcium)-treated group as compared with the control group. On the other hand, in the PCLE (calcium-free) treatment group, the effect of reducing the symptoms could not be confirmed (FIGS. 10A and 10B).
From these results, it was found that each of esterase and xylanase contributes to the disease resistance enhancing action and the plant disease controlling action.

(Purification of PaE)
P. From the culture filtrate of antarctica, "Simple biodegradable plastic decomposition using the affinity between enzymes and substrates" published in "National Institute for Agro-Environmental Sciences 2012 Research Results Information (Vol. 29)" The PaE was purified according to the "enzyme purification method" (http://www.niaes.affrc.go.jp/sinfo/result/result29/result29_42.html).
(Purification of xylanase)
P. The culture filtrate of antarctica was concentrated by ultrafiltration and replaced with 1.2 M ammonium sulfate / 50 mM sodium phosphate buffer. This solution was passed through a butyl Sepharose 4FF (GE Healthcare) column and PaE and xylanase were adsorbed on the column. The ammonium sulfate concentration in the buffer was gradually reduced to elute only xylanase from the column (PaE is still adsorbed on the column) to give xylanase without the PaE active fraction.
(Purification of PCLE)
PBSA emulsion (Showa Denko KK, EM-301) was added to Czapek-Dox liquid medium as the only carbon source, and Paraphoma genus B47-9 strain (accession number NITE P-573) was shaken and cultured. The cells of filamentous fungi were removed from the culture medium to prepare a culture filtrate. From the culture filtrate, "a simple biodegradable plastic-degrading enzyme using the affinity between the enzyme and the substrate" published in "National Institute for Agro-Environmental Sciences 2012 Research Results Information (Vol. 29)" PCLE was purified according to the purification method (http://www.niaes.affrc.go.jp/sinfo/result/result29/result29_42.html).

<Example 4>
CmCut1 which is a cutinase-like enzyme was separated and purified from the culture filtrate of the Cryptococcus magnus-related bacterium BPD1A strain (accession number NITE P-02134) which is a basidiomycete (see the preparation method described later). CmCut1 was prepared to 0.01 U / mL with an esterase titer using a buffer (20 mM Tris-HCl, pH 6.8, calcium chloride (2 mM)). As a control group, the buffer solution used for dilution (20 mM Tris-HCl, pH 6.8, calcium chloride (2 mM)) was used.
0.6 mL of the prepared enzyme solution was sprayed on the leaves of dwarf tomato (MicroTom), which is a Solanaceae plant, and kept warm while moisturizing in a closed container (humidity 100%, temperature 22 ° C.). 72 hours after spraying, 0.6 mL of pathogenic filamentous fungus (B.c.) spores (5 × 10 ⁴ spores / mL) was sprayed on each cut leaf. The state in which brown lesions were formed (may be covered with mold) was observed as a symptom, and the degree of disease was calculated based on the following formula. "Guide for conducting tests" (Japan Plant Protection Association, published in February 2003). Each test was repeated twice, using 5 cut leaves for each test plot.
Disease rate = 100 x (0 x n0 + 1 x n1 + 2 x n2 + 3 x n3 + 4 x n4) / 4N
n0: Number of leaves without lesions
n1: Number of leaves with few (several) lesions
n2: Number of leaves with lesions less than 1/4 of the leaves
n3: Number of leaves with lesions less than 1/4 to 1/2 of the leaves
n4: Number of leaves whose lesions are more than half of the leaves
N: Total number of leaves investigated FIG. 11A is a photograph on the 7th day after spraying the pathogen spores after spraying the buffer solution or CmCut1 solution. FIG. 11B shows a graph of the degree of onset calculated from the lesions observed on the 7th day after spraying the pathogen spores. As can be understood from these figures, in the 0.01 U / mL treatment group, the symptoms on the 7th day after spore spraying were alleviated as compared with the control group, and the degree of onset was reduced.

(Purification of CmCut1)
The Curyptcoccus genus magnus strain was introduced into Appl. Microbiol. Biotechnol. (2013), 97: 7679-7688 was cultured according to the method described, and the obtained culture solution was centrifuged at 7000 rpm for 5 minutes to remove bacterial cells. The obtained culture supernatant was filtered using a filter paper having a pore size of 0.45 μm (product name: Cellulose acetate C045A090C, manufactured by Advantech Co., Ltd.). And, Appl. Microbiol. Biotechnol. Affinity chromatography on PBSA emulsions was performed according to (2014), 98: 4457-4465 to purify the biodegradable plastic degrading enzyme CmCut1. It was confirmed that the purified CmCut1 was purified until it showed a single band by silver staining after SDS gel electrophoresis.

From the above, the culture of microorganisms conventionally known as a source of biodegradable plastic-degrading enzymes enhances the disease resistance of plants or enhances the disease resistance of plants at least through the activities of esterase and xylanase contained therein. It was found to control the disease.

The esterase and xylanase used in the present invention can be prepared from a microbial culture medium whose productivity has been improved in order to promote the decomposition of the biodegradable multifilm for agriculture stretched in the field, and can be mass-produced at low cost. Is something that can be done. In addition, esterase and xylanase are easily decomposed in nature, have no persistence in the environment caused by small molecule compounds and the like, and have a low environmental load. Furthermore, the composition for enhancing disease resistance of plants or controlling plant diseases of the present invention can be applied to various plants or various diseases, and is highly versatile. Therefore, the present invention has high industrial applicability.

Claims (10)

A composition for enhancing plant disease resistance or controlling plant disease, which comprises a cutinase-like enzyme and / or xylanase as an active ingredient for enhancing plant disease resistance or controlling plant disease. The composition according to claim 1 , wherein the cutinase-like enzyme and / or the xylanase is a microbial culture solution or an extract. The composition according to claim 2 , wherein the microorganism is selected from the group consisting of the genus Pseudozyma, the genus Paraphoma, and the genus Cryptococcus. The composition according to any one of claims 1 to 3 , wherein the concentration of the cutinase-like enzyme is 0.005 to 0.5 U / mL. The composition according to any one of claims 1 to 4 , which comprises both the cutinase-like enzyme and the xylanase. The composition according to any one of claims 1 to 5 , wherein the plant has a cuticle layer. The composition according to any one of claims 1 to 6 , wherein the plant is a higher plant. A method for enhancing plant disease resistance or controlling plant disease, which comprises a step of treating a plant with the composition according to any one of claims 1 to 7 . The method of claim 8 , wherein the treatment step comprises spraying the composition onto the plant. The method of claim 9 , wherein the spraying step comprises spraying 0.1 to 1 mL of the composition per leaf of the plant.

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