JP2012246227A - Soilborne disease controlling agent, and method for controlling soilborne disease using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a soilborne disease controlling agent, and a method for controlling a soilborne disease using the same.SOLUTION: This soilborne disease controlling agent contains a carbohydrate-binding module (CBM) originated in fungi. A soilborne disease can be controlled by spraying an aqueous solution containing the soilborne disease controlling agent to stems and leaves of an objective plant. In this case, the plant is preferably at least one selected from spinach, tomato, strawberry, eggplant, capsicum, green pepper and tobacco.

Description

  The present invention relates to a soil disease control agent and a soil disease control method using this control agent.

Since pathogenic fungi on plants cause damage such as reduced yields of crops and reduced quality of harvested products, their control is one of the biggest challenges in agricultural production. In the current crop production site, a large amount of fertilizer is used to cultivate a single crop over a vast area, so the damage at the time of the occurrence of a disease is particularly great. In order to avoid this damage, the use of disease control agents has become an essential technique. Diseases occurring on the stems and leaves of plants can be controlled relatively easily with the above-mentioned disease control agents, while the control of soil infectious underground diseases (hereinafter referred to as “soil diseases”) is a conventional disease control agent. Depending on the situation, it is extremely difficult. The main countermeasures against soil diseases are soil disinfection using solar heat, soil fumigant, and hot water, breeding of resistant varieties, and use of resistant rootstocks.
Of the above measures, solar heat disinfection is influenced by the weather, and it is difficult to obtain a stable effect. Soil fumigants are highly toxic and have environmental and safety issues. In addition, hot water soil disinfection is not widely used because it is affected by soil permeability and field gradients, so it has low versatility and requires water and fuel costs. The development of resistant varieties and the effects of resistant rootstocks are not yet sufficient, and the generation of pathogenic bacteria that breaks resistance becomes a problem.

In order to solve the above problems, there is a development of a disease control agent that specifically elucidates the plant pathology of pathogenic bacteria and specifically inhibits infection and development of plants based on the principle. In general, it is known that higher plants produce a resistance reaction when contacted with a pathogenic bacterium, and produce a substance exhibiting antibacterial activity against the pathogenic bacterium in a tissue around the reaction site. Substances that produce and induce such resistance substances in plants are called elicitors, and many elicitors have been isolated from phytopathogenic fungi. Representative elicitors include hepta-β-D-glucopyranoside isolated from Phytophthora megasperma f. Sp. There is icosapentaenoic acid.
Moreover, it is known that the cellulose binding domain (CBD) extracted from the cell wall of the pathogenic phytophthora parasitica nicotianae is an elicitor (Non-patent Document 1). However, this elicitor was extracted from the pathogenic bacterium itself and could not exert its effect unless it was directly injected into the plant body. Therefore, it becomes very complicated both in terms of time and work, and it causes stress for the plant body. Therefore, a large amount of plant body can be handled by a simple method, and it has high safety and low environmental load. Control agents and control methods have been desired.

Elodie Gaulin, et al., Cellulose Binding Domains of a Phytophthora Cell Wall Protein Are Novel Pathogen-Associated Molecular Patterns; The Plant Cell, Vol. 18, 1766-1777 (2006)

  The present invention has been made in view of the above-described circumstances, and the object thereof is a soil disease control agent that does not cause toxicity (including residual drugs) and appearance of drug-resistant bacteria, and soil disease control using this control agent. It is to provide a method, particularly a soil disease control agent derived from plants and microorganisms, not from pathogenic bacteria, and a soil disease control method using this control agent. Further, the present invention provides a soil disease control agent capable of exerting an effect by spraying from the outside of the plant body without performing direct administration (for example, injection) into the plant body, and a soil disease control method using this control agent. That is.

As a result of intensive studies, the present inventors have found that the occurrence of soil diseases can be prevented by spraying a carbohydrate binding module protein obtained from a microorganism onto a plant body, and basically the present invention has been completed. It was.
Thus, the soil disease control agent according to the invention for solving the above-mentioned problems is characterized by containing a fungus-derived carbohydrate binding module (CBM).
What is a carbohydrate binding module (also called “cellulose binding module”, “Carbohydrate-binding module (CBM)”, “Cellulose-binding module (CBM)”, and “module” is also called “domain”)? , One of the modules (domains) present in cellulase, and has a binding activity to carbohydrates (cellulose). Since cellulose is composed of glucose units, CBM requires sugar-protein interactions. CBMs are classified into over 50 families based on amino acid sequence homology. In terms of protein structure, there are many β-barrel structures, and it is said that aromatic amino acids (tryptophan, tyrosine, etc.) are involved in binding to sugar (stacking effect due to hydrophobicity with sugar). Modules that bind to crystalline cellulose have a flat bond surface, and aromatic amino acids are aligned there (CBM1 etc.). On the other hand, in a module that binds to amorphous cellulose or xylan, the binding surface forms a slightly concave cleft (groove) where aromatic amino acids are exposed (such as CBM6). In addition, in the module that binds to the end of the sugar chain, the cleft is at the end, and the structure is such that it is sandwiched between two aromatic amino acids (such as CBM9). These CBMs are also attracting attention for applications such as recombinant protein tags for purification and immobilization of enzymes and bacterial cells on cellulose.

CBM used in the present invention is not particularly limited, but fungi (including fungi and bacteria. Specifically, for example, cellulolytic microorganisms Clostridium genus, Ruminococcus genus, Cellulomonas genus, Streptomyces genus, Bacillus genus, Trichoderma genus, Aspergillus genus, Penicillium genus, etc.), those obtained by obtaining genes encoding CBM from these microorganisms and expressing them in heterologous expression systems, or those prepared from environmental metagenomic genes Can be used. CBM is prepared by a method disclosed in a conventionally known literature (for example, Araki, et al., Characterization of family 17 and family 28 carbohydrate-binding modules from Clostridium josui. Biosci. Biotechnol. Biochem., 73: 1028- 1032 (2009)). Further, the CBM family used in the present invention is not particularly limited, but CBM 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 17, 22, 28, 30, 37, 44 , 46, 49, 63 can be used.
A CBM solution can be prepared by cloning a gene encoding CBM and highly expressing it in a host.
Specifically, a gene encoding CBM is amplified by PCR, inserted into an expression vector, and then introduced into a host (E. coli or yeast) for expression. A recombinant microorganism having the CBM gene is cultured. In the case of Escherichia coli, the grown cells are collected by centrifugation, and ultrasonic waves are applied to destroy the cells and collect the CBM protein expressed in the cells. CBM protein can be purified using affinity chromatography with tags. In addition, since it can be expressed outside the cells when introduced into yeast, in this case, the culture solution itself can be used as a CBM solution.
The soil disease control method according to the present invention is characterized in that an aqueous solution containing the soil disease control agent is sprayed on the stems and leaves of a target plant.
The CBM concentration at the time of administration is not particularly limited, but is preferably 1 μM to 100 μM (preferably 5 μM to 80 μM, more preferably 10 μM to 50 μM).

If the CBM concentration at the time of administration is low, a large amount of the control agent may be sprayed on the target plant body, and if the CBM concentration is high, a small amount of the control agent may be sprayed on the plant body. However, when the CBM concentration is very low, the spray amount becomes too large and a large amount of water is required, and when the CBM concentration is very high, a large amount of CBM is required and the cost is increased. Therefore, the CBM concentration at the time of administration is preferably in the above range.
In addition, the target plant body is not particularly limited, and examples thereof include crops such as cereals, vegetables, fruit trees, and foliage plants. Specific examples include spinach, tomato, strawberry, eggplant, pepper, bell pepper, tobacco, banana, ginger, melon and the like.

  ADVANTAGE OF THE INVENTION According to this invention, the control agent with respect to the soil disease which was not able to cope with conventionally, and the soil disease control method using this control agent can be provided. In the present invention, CBM derived from plants and microorganisms is used instead of pathogenic bacteria, so that it is difficult to cause toxicity (including residual drugs) and appearance of drug-resistant bacteria. Furthermore, according to the present invention, it is not time-consuming as injecting the control agent directly into the plant body, but it is only necessary to spray an aqueous solution containing the control agent. . For example, by spraying a plant body with CBM, which is a kind of carbohydrate binding module, the onset of spinach wilt and tomato bacterial wilt caused by Fusarium bacteria can be remarkably suppressed. The carbohydrate binding module is a protein derived from a microorganism that is harmless to the human body, and is safer and less burdensome on the environment than conventional soil fumigants.

It is the photograph figure which showed the mode of tomato (Momotaro) bacterial wilt suppression by CBM spraying. The spray effects of BSA (A, B) and TrCBM1-CFP (C, D) were shown. It is a graph which shows the density of the pathogenic microbe which proliferated in the tomato (Momotaro) stem. It is the photograph figure which showed the mode of tomato (microtom) bacterial wilt suppression by CBM spraying. The spray effects of CFP (A, B), TrCBM1-CFP (C, D), and CjCBM3 (E, F) were shown. Appearance of tomato at the time of transplantation (A, C, E) and 4 days after the second spray of CBM (B, D, F) was shown. It is a graph which shows the dry weight of tomato (microtom) above-ground part after bacterial wilt disease onset. The above-ground dry weight on the 11th day after the second spray of CBM was shown. It is a photograph figure which shows the mode of spinach wilt suppression by CBM spraying. The spray effects of buffer solution only (A, B) and CjCBM3 (C, D) were shown. The appearance of spinach was shown during the second CBM spray (A, C) and five days after the second CBM spray (B, D). It is a graph which shows the result of having performed the gene expression analysis of the tomato (Momotaro) leaf tissue. RNA was extracted from leaves at 24 hours after CBM spraying, and DNA microarray analysis was performed. The horizontal axis shows the gene expression level in the CFP-treated strain, and the vertical axis shows the gene expression level in the TrCBM1-CFP-treated strain.

Next, embodiments of the present invention will be described with reference to the drawings. However, the technical scope of the present invention is not limited by these embodiments, and various forms can be made without changing the gist of the invention. Can be implemented. Further, the technical scope of the present invention extends to an equivalent range.
<Example 1. Preparation of protein solution>
A fungal-derived carbohydrate binding module (CBM) (TrCBM1-CFP) and a bacterial-derived CBM (CjCBM3) were prepared. As a control sample, bovine serum albumin (BSA) was used for tomato (Momotaro), cyan fluorescent protein (CFP) was used for tomato (MicroTom), and 20 mM potassium phosphate buffer was used for spinach. . Each solution was dialyzed against 20 mM potassium phosphate buffer, set to a concentration of 25 μM, and used for testing.
TrCBM1-CFP: Trichoderma reesei NBRC31329 strain genomic DNA was used as a template to amplify the region encoding cellulase Cel7A-encoding CBM1 by PCR. As primer sequences used for PCR, oligonucleotides of 5′-GGATCCCCTACCCAGTCTCACTACGG-3 ′ (SEQ ID NO: 1) and 5′-GAATTCGGGGGAGGTCAGGCACTGAGAGTAGTAAGG-3 ′ (SEQ ID NO: 2) were synthesized and used. This sequence was designed based on the nucleotide sequence published in Shoemarker et al., Nature Biotechnology 1: 691-696 (1983). A commercially available rTaq DNA polymerase was used for the PCR reaction. This amplified gene was introduced into the BamHI-EcoRI site of the expression vector pRSET-CFP to transform E. coli BL21 (DE3) strain. Under the induction with IPTG, the transformant was cultured, and the collected cells were disrupted with ultrasound to obtain a cell-free extract containing CBM. Ni-NTA agarose resin was added to this extract to adsorb CBM, and after washing, CBM was released with an imidazole solution. This was dialyzed to prepare a solution containing CBM1-CFP protein.
CjCBM3: Using the genomic DNA of Clostridium josui FERM P-9684 as a template, the region encoding CBM3 of the scaffoldin protein CipA was amplified by PCR. As primer sequences used for PCR, oligonucleotides of 5′-AAGGATCCGCAGCTGATACTGGCG-3 ′ (SEQ ID NO: 3) and 5′-AAGCTTTCAACCATTAGGTGTTGAACCA-3 ′ (SEQ ID NO: 4) were synthesized and used. This sequence was designed based on the nucleotide sequence published in Kakiuchi et al., Journal of Bacteriology 180: 4303-4308 (1998). This amplified gene was introduced into the BamHI-HindIII site of the expression vector pQE30, and Escherichia coli JM109 strain was transformed. Under the induction with IPTG, the transformant was cultured, and the collected cells were disrupted with ultrasound to obtain a cell-free extract containing CBM. Ni-NTA agarose resin was added to this extract to adsorb CBM, and after washing, CBM was released with an imidazole solution. This was dialyzed to prepare a solution containing CjCBM3 protein.

<Example 2. Tomato (Momotaro) Cultivation and Disease Control Test Method>
Tomato seeds (Momotaro) were sown in cell trays filled with culture soil and grown in a glass greenhouse. In addition, 1 ward was set to 7 shares. 3 mL of CBM solution was sprayed on seedlings with a plant height of about 10 cm. Seven days after the spray treatment, the seedlings were transplanted into pots filled with Ralstonia solanacearum-contaminated soil (10 ⁶ cfu / g), and cultivation was continued in the greenhouse. Three days after transplantation, 3 mL of CBM solution was sprayed again. The disease was investigated 4 days after the second spray. Furthermore, the density (cfu / g) of pathogenic bacteria grown in the stem was measured.
The gene expression situation after CBM spray treatment was analyzed. In the same manner as described above, seedlings were grown in a cell tray to a plant height of about 10 cm and sprayed with 1 mL of protein solution. Twenty-four hours after the spray treatment, leaves were collected, and RNA was extracted therefrom to prepare a sample. Samples were analyzed with a DNA microarray.

<Example 3. Tomato (Microtom) Cultivation and Disease Control Test Method>
Tomato seeds (microtomes) were sown in a cell tray packed with vermiculite, and the seedlings were grown by controlling them at 30 ° C. in an artificial weather apparatus while giving liquid fertilizer. In addition, 1 ward 6 shares. 3 mL of CBM solution was sprayed on seedlings with a plant height of about 2 cm. Three days after spraying, the seedlings were transplanted into pots filled with bacterial wilt contaminated soil (10 ⁶ cfu / g), and cultivation was continued in the same meteorological apparatus. Three days after transplantation, 3 mL of CBM solution was sprayed again. From the second spray to the fourth to eleventh days, the progression of symptoms was investigated and classified into the following six stages of disease index. Disease index (0: 0% wilted leaf ratio, 1: 1-25%, 2: 26-50%, 3: 51-75%, 4: 76-99%, 5: 100%). Moreover, the control value was calculated by the following formula using the disease index.
Control value = {1− (average disease incidence index of treatment group / average disease incidence index of control group)} × 100.

<Example 4. Spinach cultivation and disease control test method>
Spinach seeds (okame) were sown in cell trays filled with culture soil, and the seedlings were grown at 26 ° C. in an artificial meteorological device. In addition, it was set to 13 shares in 1 district. Four days after sowing, seedlings were sprayed with 3 mL of CBM solution. Three days after the spray treatment, Fusarium oxysporum f. Sp. Spinaciae GF960 (10 ⁴ cfu / g) was inoculated. Three days after inoculation, 3 mL of CBM solution was sprayed again. Five days after the second spray, the progression of symptom was investigated and classified into the following five stages of disease index. Disease index (0: healthy, 1: healthy above ground but browned root surface, 2: healthy above ground but browned root conduit, 3: wilt above ground, 4: dead). Furthermore, the control value was calculated using the disease index.

<Result>
Inhibitory effect of bacterial wilt on tomato (Momotaro) In the control group, 86% of tomato strains developed bacterial wilt and died. On the other hand, the death rate of the tomato strain sprayed with TrCBM1-CFP was only 29%, and a high disease suppression effect was observed (FIG. 1). Moreover, when the pathogenic bacteria in the stem were compared, the TrCBM1-CFP treatment group showed a reduction to about 1/3 of the control group (FIG. 2). From the above results, it became clear that the tomato strain became resistant to bacterial wilt by TrCBM1-CFP spray treatment, and the infection and growth of pathogenic bacteria in the tomato body were suppressed.

Inhibitory effect of bacterial wilt in tomato (microtom) In the control group, 100% of tomato strains died 4 days after the second CBM spray (FIG. 3). On the other hand, in the TrCBM1-CFP treatment group, the disease was suppressed, the disease index after 4 days of the second spray was 1.3 (control value 74), and the disease index after 11 days of the second spray was 3.3 (34) (FIG. 3, Table 1). Furthermore, in the CjCBM3 treatment group, no pathogenic strain appeared 4 days after the second spray (control value 100), and the disease index remained at 0.8 (control value 84) even 11 days after the second spray (FIG. 3, Table 1). ). In the CBM-treated group, the dry weight after 11 days of the second spray was significantly larger than that in the control group (FIG. 4). Thus, it has been clarified that the treatment of both proteins exhibits a high control effect against bacterial wilt, which is a soil-borne bacterial disease that is difficult to control.

Control effect against spinach wilt
CjCBM3 spray remarkably suppressed the onset of spinach wilt (FIG. 5). As shown in Table 2, the disease index of the untreated group was 2.2, but the CjCBM3 treated group was 1.0 (control value 55). From this result, it became clear that foliar spray of CjCBM3 is also effective for soil-borne fungal diseases.

Gene expression analysis
Microarray analysis of gene expression in TrCBM1-CFP sprayed tomato (Momotaro) strains revealed that expression of defense-related genes such as ethylene-responsive genes and stress-responsive genes was remarkably induced (Table) 3, FIG. 6). This result suggests that just spraying TrCBM1-CFP onto plant leaves induces disease and stress resistance throughout the plant body.
Table 3 below shows an example of genes whose leaves were collected 24 hours after TrCBM1-CFP spraying, RNA was extracted, and DNA microarray analysis was performed. Indicated.

As described above, according to the present embodiment, it is possible to provide a control agent for bacterial wilt and wilt disease, which are soil diseases for which there have been few methods for effectively dealing with it, and a soil disease control method using this control agent. It was. In this embodiment, since CBM derived from plants and microorganisms is used instead of pathogenic bacteria, toxicity (including residual drugs) and appearance of drug-resistant bacteria are unlikely to occur. Furthermore, in this embodiment, it is not time-consuming as injecting into a plant body, and it is only necessary to spray a control agent, so that an easy control method can be provided. For example, by spraying CBM, which is a kind of carbohydrate binding module, on plants, the onset of spinach wilt disease and tomato bacterial wilt caused by Fusarium bacteria could be remarkably suppressed. CBM is a protein derived from microorganisms that are harmless to the human body, and can provide a soil disease control agent that is safer and less burdensome on the environment than conventional soil fumigants.

Claims (6)

A soil disease control agent comprising a saccharide-binding module (CBM) derived from a fungus. The carbohydrate binding module (CBM) is prepared from the cellulolytic microorganisms Clostridium genus, Ruminococcus genus, Cellulomonas genus, Streptomyces genus, Bacillus genus, Trichoderma genus, Aspergillus genus, Penicillium genus, or CBM from these microorganisms. The soil disease control agent according to claim 1, wherein the agent is selected from those obtained by obtaining a gene coding for and expressing in a heterologous expression system, or those prepared from metagenomic genes in the environment. The carbohydrate binding module (CBM) consists of CBM 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 17, 22, 28, 30, 37, 44, 46, 49, 63 The soil disease control agent according to claim 1 or 2, which is one or more selected from the group. It is a soil disease control agent as described in any one of Claims 1-3, Comprising: The target plant body consists of a spinach, a tomato, a strawberry, eggplant, a pepper, a bell pepper, a tobacco, a banana, ginger, and a melon. A soil disease control agent, which is at least one selected from the group consisting of: The soil disease control method characterized by spraying the aqueous solution containing the soil disease control agent as described in any one of Claims 1-3 to the stalk and leaf of the target plant body. The soil disease according to claim 5, wherein the plant body is at least one selected from the group consisting of spinach, tomato, strawberry, eggplant, pepper, pepper, tobacco, banana, ginger and melon. Control method.