WO2017204288A1 - Composition for enhancing disease resistance of plants or controlling plant disease, and method for using same - Google Patents

Abstract

The purpose of the present invention is to provide a novel use for biodegradable-plastic-degrading enzymes and a culture of microorganisms including the same. Esterase and/or xylanase can be used to enhance the disease resistance of plants or control plant disease.

Description

Composition for enhancing plant disease resistance or controlling plant disease and methods for using them

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

Plants are equipped with a biological defense system that conforms to animal immunity. When this function is activated, the plant activates various defense systems and becomes a plant that is unlikely to become diseased. In recent years, in the development of bactericides, from the viewpoint of environmental protection and avoidance of drug-resistant bacteria, there has been an active search for drugs that use these defense systems to control diseases. These drugs (1) have a preventive effect against multiple pathogens (wide spectrum of action), (2) very low incidence of resistant bacteria, (3) long-term control effect (4) It has the feature that there is little direct influence 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, only a limited number of compounds have been put to practical use. In addition, all the drugs marketed in Japan target rice, but similar drugs are required for various crops.

Also, the surface layer of the plant is covered with a cuticle layer and wax in order to protect the body from external stresses, especially osmotic stress caused by transpiration and rainfall, invasion of pathogenic bacteria, and pest damage. An enzyme that hydrolyzes cutin (fatty acid polyester) contained in this cuticle layer is called cutinase.

Recently, a technique for rapidly degrading biodegradable plastic agricultural materials after use with enzymes obtained from plant-resident yeast and filamentous fungi has been developed (Patent Documents 1 and 2). In addition, techniques for mass-producing a plurality of enzymes have 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. And when a plant is treated with a culture filtrate containing these biodegradable plastic-degrading enzymes derived from filamentous fungi or yeast at a high concentration, a phenomenon has been found that the plant dies. At this time, the cuticle layer is thin and easily infected with phytopathogenic fungi. Enzymes capable of decomposing part of the cuticle layer, such as biodegradable plastic degrading enzymes, are used as herbicides to control harmful plants. It can be used (Patent Document 5). Moreover, it has been found that a culture solution containing a biodegradable plastic-degrading enzyme derived from yeast Pseudozyma antarctica also contains xylanase (hemicellulose-degrading enzyme) (Non-patent Document 2). However, no biodegradable plastic-degrading enzyme derived from yeast has actually been confirmed to have a cactinolytic activity. In addition, a method for enhancing plant disease resistance and controlling plant diseases with biodegradable plastic degrading enzymes has not been known.

Patent No. 4915593 Patent No. 5082125 Patent No. 5849297 International Publication No. 2014/109360 JP 2014-129287 A

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

As a result of intensive studies to solve the above problems, the present inventors have surprisingly found that a culture of a microorganism used as a biodegradable plastic degrading enzyme is an esterase and / or contained therein. Through the action of xylanase, the present inventors have found that the present invention has an effect of suppressing the infection of pathogenic bacteria and an effect of suppressing germination of pathogenic bacteria, and increases the expression of stress resistance genes in plants.
That is, the present invention provides a plant disease resistance enhancing or plant disease controlling composition described below, and a method for enhancing plant disease resistance or controlling plant diseases using them. is there.
[1] A composition for enhancing plant disease resistance or controlling plant diseases comprising esterase and / or xylanase.
[2] The composition according to [1], wherein the esterase contains a biodegradable plastic degrading enzyme.
[3] The composition according to [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 [1] to [3], wherein the esterase and / or the xylanase is a microorganism culture solution or an extract.
[5] The microorganism is a genus Pseudozyma, a Paraforma, a Cryptococcus, a Mucor, a Humicola, a Thermomyces, a Taromomis, or a Taromomis, The genus Chaetomium, the genus Torula, the genus Sporotricum, the genus Malblanche, the genus Alternaria, the genus Cladosporium, the genus Penicillium, Penicillium ) Genus, Pseudomonas genus, Ba Teroidesu (Bacteroides) genus, and is selected from the group consisting of Acidovorax (Acidovorax) genus composition according to the above [4].
[6] The composition according to any one of [1] to [5], wherein the esterase concentration is 0.005 to 0.5 U / mL.
[7] The composition according to any one of [1] to [6], comprising both the esterase and the xylanase.
[8] The composition according to any one of [1] to [7], wherein the plant has a cuticle layer.
[9] The composition according to any one of [1] to [8], wherein the plant is a higher plant.
[10] A method for enhancing plant disease resistance or controlling plant disease, comprising the step of treating the plant with the composition according to any one of [1] to [9].
[11] The method according to [10], wherein the treatment step includes a step of spraying the composition onto the plant.
[12] The method according to [11], wherein the spraying step includes a step of spraying 0.1 to 1 mL of the composition per leaf of the plant.

According to the present invention, the disease resistance of plants is enhanced by esterase, which is a biodegradable plastic-degrading enzyme contained in a culture of microorganisms, and / or xylanase contained in the culture. Plant diseases can be controlled. Plant disease resistance enhancers have been studied for a long time, but only three types of drugs targeting rice are known as products that have become domestic products. However, the disease resistance enhancing action or disease controlling action by the esterase and / or xylanase of the present invention can be used not only for gramineous plants but also for a wide variety of crop species such as tomato, syleusona, and tobacco, and has versatility. high. In addition, since the esterase and xylanase which are the active ingredients of the present invention are not low molecular weight compounds known as conventional disease resistance enhancers but are proteins, they are easily decomposed in the natural world. There is no persistence in the environment caused by compounds, and the environmental load is low.

A photograph showing changes in leaf symptom. The graph which shows the symptom change of a leaf. Micrograph of pathogenic spore. Graph of germination rate of pathogenic fungus spores. Graph of DNA amount derived from pathogenic bacteria. Photomicrograph of leaves containing pathogenic bacteria. Graph of stress resistance genes in plants. The graph of the reactive oxygen species response gene of a plant. Leaf photo. Disease severity graph. Leaf photo. Disease severity graph. Leaf photo. Graph of lesion diameter. Leaf photo. Disease severity graph. Leaf photo. Disease severity graph.

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, “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. For example, they are used as biodegradable plastic degrading enzymes. The enzyme may be used.

As used herein, the term “biodegradable plastic degrading enzyme” refers to an enzyme having an activity of degrading biodegradable plastic. Any enzyme can be used in the composition of the present invention without limitation as long as it has an activity of degrading biodegradable plastics. For example, cutinase or cutinase-like enzyme may be employed. The activity of degrading the biodegradable plastic is measured by measuring the degradation activity of polybutylene succinate-co-adipate (PBSA) emulsion, or PBSA, polybutylene succinate, polybutylene adipate terephthalate, polylactic acid, etc. This can be determined by measuring the degradation activity of the biodegradable plastic film.

The “xylanase” described in the present specification refers to an enzyme having an activity of decomposing xylan into xylose, and can decompose hemicellulose in the plant cell wall. In the composition of the present invention, various xylanases can be used without limitation as long as they have an activity of degrading 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. Also good. The microorganism that produces the esterase and / or the xylanase is not particularly limited, and examples thereof include the genus Pseudozyma, the genus Paraforma, the genus Cryptococcus, the genus Mucor, and the Humicola. (Humicola), Thermomyces, Talaromyces, Ketomium, Torula, Sporotrichum, Malbrana The genus Spodium, the genus Penicillium, Pequillomyce (Paecilomyces) genus Pseudomonas (Pseudomonas) genus Bacteroides (Bacteroides) genus, and it may be a microorganism selected from the group consisting of Acidovorax (Acidovorax) genus.

The biodegradable plastic degrading enzyme as the esterase is preferably Pseudozyma antarctica (for example, GB-4 (1) W strain, GB-4 (0) -HPM7 strain (incorporated administrative agency, Product Evaluation Technology Infrastructure Organization, Patent Microorganism Deposit) Yeast deposited at the Center; Deposit No. NITE BP-02238) and OMM62-2 strain (Yeast deposited at the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation, Accession No. NITE BP-02239) Esterase (PaE), Paraphoma-related fungus cutinase-like enzyme (PCLE), or cutinase-like enzyme 1 (CmCut1). PCLE is Paraphoma such as Paraphoma genus B47-9 strain (filamentous fungus deposited at the National Institute of Technology and Evaluation of Microorganisms of the National Institute of Technology and Evaluation; Accession Number NITE P-573; refer to Japanese Patent No. 5082125 if necessary) CmCut1 is a Cryptococcus magnus such as Cryptococcus magnus genus BPD1A strain (yeast deposited with the Patent Microorganism Depositary of the National Institute of Technology and Evaluation of the Independent Administrative Institution; Accession Number NITE P-02134) Or it is an enzyme produced by its related bacteria.

The concentration of the esterase in the composition of the present invention can be appropriately adjusted according to the type of plant to be applied and the type of the pathogen. The esterase concentration 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 esterase titer shown here is determined by measuring the decrease in turbidity of polybutylene succinate-co-adipate (PBSA) emulsion (Showa Denko EM-301), a biodegradable plastic. It has been decided. The titer when the value of OD660nm was decreased by 1 per minute was defined as 1U. As a buffer solution for measuring enzyme activity, for example, a Tris-HCl 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 plant to be applied and the type of the pathogenic fungus. The concentration of the xylanase may be, for example, 0.001 to 5.0 U / mL, and preferably 0.005 to 1.0 U / mL.
The titer of xylanase shown here is the amount of xylose produced when xylan is decomposed, Agric. Biol. Chem. (1980), 44 (12), 2943-2949, measured by a modified method of the Somogi-Nelson method. The titer that produces 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 action mechanisms in combination, a synergistic plant disease resistance enhancing action and / or plant disease controlling action is achieved. When the esterase and the xylanase are used in combination, the isolated and purified esterase and xylanase may be blended in the same composition, but a microorganism culture solution or extract containing these enzymes from the beginning is used. This is convenient. Alternatively, a composition containing only the esterase and a composition containing only the xylanase may be used simultaneously or sequentially.

The “enhancement of disease resistance of a plant” described in the present specification refers to the induction of a situation in which infection is not established or disease symptoms are reduced even when any pathogen contacts the plant. “Plant disease resistance enhancement” means, for example, elevation of injury response genes such as PIN2 (proteinase inhibitor 2) and LapA1 (leucine aminopeptidase A1), and disease response genes such as PR4 (pathogenesis related 4) and PRB1b. In some cases, such as the release of reactive oxygen species, an enhancement of the physiological response of a plant to a disease can be an indicator. In addition, “plant disease control” described in the present specification refers to suppressing symptom changes in plants as a result regardless of the mechanism of action. Examples of the action mechanism for controlling plant diseases include, but are not limited to, changes in physiological responses of plants as described above, changes in symbiotic microflora, or attenuation of pathogenic bacteria. Absent.

The composition of the present invention can be used for various plants because it exhibits non-specific plant disease resistance enhancing action and plant disease controlling action regardless of the kind of plant or disease. The composition of the present invention may be used, for example, for plants having a cuticle layer, or higher plants such as a seed plant (a gymnosperm or angiosperm) and a fern plant. In addition, the composition of the present invention can be used for various crops, for example, mallow, akaza, cruciferous, iridaceae, iridaceae, gramineous, saccharidaceae, araliaceae, cucurbitaceae, cypressaceae, asteraceae, walnutaceae. , Mulberry, poppy, primrose, primaceae, taro, cactaceae, perilla, cypridaceae, ginger, waterlily, violet, sericaceae, gypsaceae, azalea, camellia, euphorbiaceae, eggplant Family, urchinaceae, rose family, antaceae family, convolvulaceae family, sorghum family, grape family, beech family, button family, matabidae family, legume family, mandarin family, genus magnolia family, saxifrage family, lily family, orchid family, agave family, And you may use with respect to the crops which belong to the family selected from the group which consists of Gentianaceae. And the composition of this invention can enhance the resistance with respect to various diseases, such as mold disease, bacterial disease, and viral disease, or can control such various diseases.

The composition of the present invention may further contain other arbitrary components as long as the disease resistance enhancing action or the plant disease control action of the plant is not inhibited. 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 plant disease controlling action of the plant.

In one aspect, the present invention also relates to a method for enhancing plant disease resistance or controlling plant diseases, comprising the step of treating a plant with a plant disease resistance enhancing composition or plant disease controlling composition. Related. As a method for the treatment, various methods can be used without limitation as long as it results in enhancing the disease resistance of the plant or controlling the disease of the plant. For example, the treatment step may include a step of spraying the composition onto the plant. Although the application site | part of the said composition is not specifically limited, For example, the leaf, stem, fruit, etc. of the said plant may be sufficient. The application amount of the composition can be appropriately adjusted according to the application method and application site, and for example, 0.1 to 1 mL of spray per leaf of the plant may be used.

Hereinafter, the present invention will be specifically described by way of 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) Antarctica (GB-4 (1) W strain) was precultured in YM medium (yeast extract malt extract medium) shown in Table 1.

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

In order to prevent the culture fluid from flowing out due to the formation of high bubbles, a 50-fold diluted solution of an antifoaming agent (trade name “Shin-Etsu Silicone KM-72F”, manufactured by Shin-Etsu Chemical Co., Ltd.) is intermittently used using an anti-foam sensor. About 40 mL was added by 72 hours after the start of culture.
In addition, the pH drop due to ammonium ion consumption in the medium is detected by a sensor, and ammonia water is automatically added to the medium as an alkali adjustment solution to adjust the pH to 6.0 in order to add a nitrogen source and adjust the pH. did. From 24 hours after the start of the culture, a fed-batch medium having the composition shown in Table 3 was fed at a rate of 0.5 L / d. Finally, the culture solution after 72 hours of cultivation was filtered through CELLULOSE ACEDATE filter paper (manufactured by ADVANTEC) having a pore size of 0.45 μm to prepare a culture filtrate. This culture filtrate was used in the following tests. P.P. Even if another antarctica strain (GB-4 (0) -HPM7 strain or OMM62-2 strain) is used, the culture filtrate should be prepared in the same manner as when the GB-4 (1) W strain is used. And could be used as well.

(2) Spray treatment on cut leaves Antarctica culture filtrate (esterase titer: 8.0 U / mL) was diluted with 20 mM Tris-HCl buffer (pH 8.8) to obtain 0.01 U / mL, 0.1 U / mL, 1.0 U / mL. A solution was prepared. Each leaf cut from dwarf tomato (MicroTom) was sprayed with 0.6 mL of each solution of 0.01 U / mL to 8.0 U / mL and kept warm in a sealed container (humidity) 100%, temperature 22 ° C.). The control filtrate was sprayed with a culture filtrate in which the enzyme was inactivated by autoclaving. 72 hours after spraying (3 days later), spores (5 × 10 ⁴ spores / mL) of pathogenic filamentous fungi (Gray mold fungus (Botrytis cinerea, B.c.)) were applied to each cut leaf. Sprayed with 0.6 mL each. Five cut leaves were used for each treatment group, and the state of the leaves was observed on the 10th day after the enzyme treatment, that is, 7 days after the spore treatment. Symptoms that form brown lesions (may be covered with mold), no obvious symptoms, but yellowing of leaves that are green in green color, both symptom and yellowing The state that was not able to be evaluated was evaluated as unchanged. FIG. 1A shows a photograph of a cut leaf that was sprayed with a pathogenic fungus spore after each enzyme treatment, and FIG. 1B shows the ratio of the observed cut leaf state (no change, yellowing, or disease symptoms). A graph is shown. As can be seen from these figures, the 1.0 U / mL and 8.0 U / mL treatment groups promoted infection efficiency and promoted disease symptoms, whereas the 0.1 U / mL treatment group promoted the same symptoms as the control group. In the 0.01 U / mL treatment group, the symptom on day 10 was reduced compared to the control group.

(3) Germination rate of pathogenic bacteria Mold spores and mycelia on the leaf surface on the second day after inoculation with pathogenic bacteria (Bc) were stained with trypan blue. Four cut leaves were used for each treatment group and observed with a microscope. FIG. 2A shows an optical microscope observation photograph of mold spores on the leaf surface two days after spraying the pathogenic fungus spores after each enzyme treatment, and FIG. 2B is a graph of the average germination rate of the observed mold spores (error range is Standard error calculated from 4 tests). Here, the different alphabets (a, b, and c) in FIG. 2B are significantly different in germination rates among the treatment groups as a result of a significant difference 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 each treatment group. Specifically, germination of pathogenic bacteria spores is significantly suppressed in the 0.01 U / mL, 0.1 U / mL, and 1.0 U / mL treatment groups as compared to the control group and 8.0 U / mL treatment group. (FIGS. 2A and 2B). In the control group and the 8.0 U / mL treatment group, it was observed that the germs spores germinated and the germ tubes spread on the leaf surface. In particular, in the 8.0 U / mL treatment group, the germination rate was significantly higher than that in the control group.

(4) Abundance of pathogenic bacteria in leaves DNA was extracted from leaves on the third day after spore inoculation with pathogenic bacteria (Bc), and the DNA amount of pathogenic bacteria was quantified by qPCR. Five cut leaves were used for each treatment section. Fig. 3 shows the results of analysis using the β-tubulin gene as an index, that is, the average value of the relative amount of β-tubulin gene relative to the correction with the amount of Actin gene in tomato (the error range is calculated from 5 tests). Standard error). As a result, the amount of pathogenic bacteria is remarkably high in the 1.0 U / mL and 8.0 U / mL treatment sections, and it can be confirmed that the pathogenic bacteria are infected. On the other hand, in the 0.01 U / mL and 0.1 U / mL treatment groups, it was confirmed that the pathogenic bacteria were clearly suppressed as compared with the control group.

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

(5) Plant Response Analysis RNA is extracted from the leaves on the third day after inoculation with pathogenic bacteria (B. c.), And various stress resistance-related genes of plants (PIN2 and LapA, which are injury response genes) are analyzed by qRT-PCR. In addition, the expression levels of disease-responsive genes PR4 and PRB1b) were examined (corrected with tomato Actin gene dosage). Three cut leaves were used for each treatment section. FIG. 5 shows the average value of the relative expression level of various stress resistance-related genes with respect to the actin gene of tomato (the error range is a 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 detect not only stress resistance-related genes but also reactive oxygen species response genes because they release active oxygen when the cuticle layer destruction or elicitor is detected. Changes in the expression level of reactive oxygen species responsive genes were examined by qRT-PCR using the extracted RNA. Peroxidase (POD) and superoxide for the tomato Actin gene, especially with 0.01 U / mL culture filtrate treatment It was confirmed that the average value of the relative expression level of dismutase (SOD) (the error range is a standard error calculated from three tests) was increased (FIG. 6).

(6) Decomposition activity of cutin It has been confirmed that fatty acids having carbon chain lengths of 16 and 18 are extracted from the cuticular 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 of the plant surface layer, P.P. The degradability of the 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 degradation product of cutin, was detected in the reaction solution. From this, P.I. It was found that the culture solution of antarctica has an activity to release the cutin monomer from the plant.

<Example 2>
(1) Effects on monocotyledonous plants and their pathogens Antarctica culture filtrate was diluted as in Example 1. An esterase titer of 0.01 U / mL was sprayed on the leaves of oats, which are gramineous plants, and kept warm in a sealed container (humidity 100%, temperature 22 ° C.). . The control filtrate was sprayed with a culture filtrate in which the enzyme was inactivated by autoclaving. After 72 hours of spraying, the inoculum was inoculated into a culture solution (OD610: 0.65) of a pathogenic bacterium, B. gonorrhoeae, which infects a monocotyledon oat. The same test was repeated twice using 4 cut leaves for each treatment group. A state in which an elliptical brown spot (sickness spot) spreads vertically was observed as a symptom. In addition, the lesion area was observed, and the disease severity was calculated based on the following formula (for the disease severity calculation method, “Field test method for fungicides such as rice and wheat” (Japan Plant Protection Association, 2004) Issue in March).
Disease severity = 100 x (0 x n0 + 0.5 x n1 + 1 x n2 + 2 x n3 + 3 x n4 + 4 x n5) / 4N
n0: The number of leaves without lesions n1: The number of leaves whose lesions are 1/8 or less of the leaves n2: The number of leaves whose lesions are around 1/4 of the leaves n3: The number of leaves 1 / of the leaves The number of leaves that are around 2 n4: The number of leaves whose lesion is around 3/4 of the leaf n5: The number of leaves whose lesion is 7/8 or more of the leaf N: The total number of leaves investigated Figure 7A A photograph of the third day after inoculation with pathogenic bacteria after each enzyme treatment is shown, and FIG. 7B shows a graph of disease severity calculated from lesions observed on the third day after inoculation with pathogenic bacteria. As can be understood from these figures, in the 0.01 U / mL treatment group, the symptom on the third day of inoculation was reduced as compared with the control group, and the disease severity was reduced.

(2) Brassicaceae plants and their effects on pathogenic bacteria. Antarctica culture filtrate was diluted as in Example 1. A solution of 0.01 U / mL of esterase titer was sprayed on rosette leaves of Arabidopsis thaliana, which was a cruciferous plant, and kept warm in a sealed container (humidity 100%, temperature 22 ° C. ). The control filtrate was sprayed with a culture filtrate in which the enzyme was inactivated by autoclaving. 72 hours after spraying, spores (5 × 10 ⁴ spores / mL) of pathogenic filamentous fungi (gray mold fungus) were sprayed. The same test was repeated twice using 8 cut rosette leaves for each treatment section. The condition of brown lesions was observed as a symptom, and the disease severity was calculated based on the following formula. (For the calculation method of disease severity, "Guidelines for conducting a new pesticide practical application test" (Japan Corporate (See Plant Protection Association, issued February 2003)).
Disease severity = 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 leaves n3: lesions 1/4 of leaves Number of leaves that are less than ½ n4: Number of leaves with lesions greater than or equal to ½ of leaf N: Total number of leaves investigated Figure 8A shows the third day after spraying with pathogenic fungi after each enzyme treatment FIG. 8B shows a graph of disease severity calculated from lesions observed on the third day after spraying the pathogenic fungus spores. As can be understood from these figures, in the 0.01 U / mL treatment group, the symptom on the seventh day after spore spraying was reduced compared to the control group, and the disease severity decreased.

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

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

0.6 mL of each prepared enzyme solution was sprayed onto oat leaves, which are grasses, and kept warm in a sealed container (humidity 100%, temperature 22 ° C.). 72 hours after spraying, the seedling was inoculated by immersion in a culture solution (OD610: 0.65) of a Japanese wilt disease that infects monocotyledonous plants. Six cut leaves were used for each treatment section. FIG. 10A shows a photograph of the fourth day after inoculation of pathogenic bacteria in various enzyme treatments, and FIG. 10B shows a graph of disease severity calculated from lesions observed on the fourth day after inoculation of pathogenic bacteria. As can be understood from these figures, in the PaE-treated group, the xylanase-treated group, and the PCLE (with calcium) -treated group, the symptoms on the fourth day of inoculation were reduced compared to the control group. On the other hand, in the PCLE (no calcium) treatment section, the symptom reduction effect could not be confirmed (FIGS. 10A and 10B).
From these results, it was found that each of esterase and xylanase contributed to the disease resistance enhancing action and the plant disease controlling action.

(PeE purification)
P. simple biodegradable plastic degradation using the affinity of enzyme and substrate published in "Agricultural Environment Technology Laboratory 2012 Research Results Information (Vol. 29)" 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. While the antarctica culture filtrate was concentrated by ultrafiltration, it was replaced with 1.2 M ammonium sulfate / 50 mM sodium phosphate buffer. This solution was passed through a butyl sepharose 4FF (GE Healthcare) column to adsorb PaE and xylanase to the column. The ammonium sulfate concentration in the buffer was gradually decreased, and only xylanase was eluted from the column (PaE was still adsorbed on the column) to obtain a xylanase containing no PaE active fraction.
(Purification of PCLE)
PBSA emulsion (Showa Denko KK, EM-301) was added to Czapek-Dox liquid medium as the sole carbon source, and Paraphoma-related bacteria B47-9 (accession number NITE P-573) was cultured with shaking. Filamentous fungi were removed from the culture solution to prepare a culture filtrate. From the culture filtrate, a simple biodegradable plastic-degrading enzyme using the affinity between the enzyme and the substrate is published in "Agricultural Environmental Technology Research Institute 2012 Research Results Information (Vol. 29)". PCLE was purified according to “Purification method” (http://www.niaes.affrc.go.jp/sinfo/result/result29/result29 — 42.html).

<Example 4>
CmCut1, which is a cutinase-like enzyme, was isolated and purified from the culture filtrate of the basidiomycete Cryptococcus magnus strain BPD1A strain (Accession No. NITE P-02134) (see the preparation method described later). CmCut1 was prepared to 0.01 U / mL with an esterase titer using a buffer solution (20 mM Tris-HCl, pH 6.8, calcium chloride (2 mM)). As a control, the buffer 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 solanaceous plant, and kept warm in a sealed container (humidity 100%, temperature 22 ° C.). 72 hours after spraying, each cut leaf was sprayed with 0.6 mL of pathogenic filamentous fungus (Bc) spores (5 × 10 ⁴ spores / mL). Observed as a disease symptom that brown lesions are formed (may be covered with mold), and calculated the disease severity based on the following formula "Guide for conducting tests" (see Japan Plant Protection Association, published in February 2003)). Each test was repeated twice, using 5 cut leaves for each test section.
Disease severity = 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 leaves n3: lesions 1/4 of leaves Number of leaves that are less than ½ n4: Number of leaves with lesions greater than or equal to ½ of leaves N: Total number of leaves examined FIG. 11A shows the pathogenic fungal spores after spray treatment with buffer or CmCut1 solution Photo 7th day after spraying. FIG. 11B shows a graph of disease severity calculated from lesions observed on the seventh day after nebulization of pathogenic fungi. As can be understood from these figures, in the 0.01 U / mL treatment group, the symptom on the seventh day after spore spraying was reduced compared to the control group, and the disease severity decreased.

(Purification of CmCut1)
A strain of the genus Cryptcoccus genus bacterium, Magnus strain, was prepared by using Appl. Microbiol. Biotechnol. (2013), 97: 7679-7688, and the resulting culture was centrifuged at 7000 rpm for 5 minutes to remove the cells. The obtained culture supernatant was filtered using a filter paper having a pore diameter of 0.45 μm (product name: Cellulose acetate C045A090C, manufactured by Advantech Co., Ltd.). And Appl. Microbiol. Biotechnol. (2014), 98: 4457-4465, affinity chromatography on PBSA emulsion was performed to purify CmCut1, which is a biodegradable plastic degrading enzyme. It was confirmed that the purified CmCut1 was purified until a single band was shown by silver staining after SDS gel electrophoresis.

From the above, the culture of microorganisms conventionally known as a biodegradable plastic-degrading enzyme source has enhanced plant disease resistance or the plant's disease resistance through at least 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 microorganism culture solution whose productivity has been improved to promote the degradation of agricultural biodegradable multifilms stretched in the field, and is inexpensively mass-produced. Is something that can be done. In addition, esterase and xylanase are easily decomposed in nature, have no persistence in the environment caused by low molecular weight compounds, and have a low environmental load. Furthermore, the plant disease resistance enhancing or plant disease controlling composition 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.

[Supplement under rule 26 15.06.2017]

Claims (12)

1. A composition for enhancing plant disease resistance or controlling plant diseases comprising esterase and / or xylanase.
2. The composition according to claim 1, wherein the esterase comprises a biodegradable plastic degrading enzyme.
3. The composition according to claim 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 claims 1 to 3, wherein the esterase and / or the xylanase is a microorganism culture solution or an extract.
5. The microorganism may be Pseudozyma, Paraforma, Cryptococcus, Mucor, Humicola, Thermomyces, Taromemis, Taromemis, Taromomis, ), Torula, Sporotrichum, Malblancea, Alternaria, Cladosporium, Penicillium, c, Pequillomyces, e Pseudomonas genus, bacte Idesu (Bacteroides) genus, and is selected from the group consisting of Acidovorax (Acidovorax) genus composition of claim 4.
6. The composition according to any one of claims 1 to 5, wherein the esterase concentration is 0.005 to 0.5 U / mL.
7. The composition according to any one of claims 1 to 6, comprising both the esterase and the xylanase.
8. The composition according to any one of claims 1 to 7, wherein the plant has a cuticle layer.
9. The composition according to any one of claims 1 to 8, wherein the plant is a higher plant.
10. A method for enhancing plant disease resistance or controlling plant diseases, comprising the step of treating plants with the composition according to any one of claims 1 to 9.
11. The method according to claim 10, wherein the treatment step includes a step of spraying the composition onto the plant.
12. The method according to claim 11, wherein the spraying step includes a step of spraying the composition from 0.1 to 1 mL per leaf of the plant.

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