JP6739728B2 - Control agent for bacterial diseases of gramineous plants, control method and seed coated with the control agent - Google Patents

Description

The present invention relates to a bacterial disease control agent effective for controlling bacterial diseases of grasses, a method for controlling bacterial diseases of grasses, and seeds of grasses coated with the controlling agent.

In modern agriculture, pest control technology centered on so-called chemical pesticides has been developed in order to efficiently secure food. However, as a result of excessive use of chemical pesticides for many years, problems such as disturbance of the ecosystem, food safety due to residual pesticides, health damage to farmers using chemical pesticides, etc. are highlighted, and safety and safety are considered. Therefore, conversion to pesticides with low toxicity and low persistence is required, and further reduction and pesticide-free efforts are being demanded.

In such a trend, pest control technology (for example, microbial control agent) suitable for environmental conservation type agriculture in consideration of reduction of environmental load due to use of pesticides has been attracting attention. "Microbial control agents" are formulations that protect crops from damage such as pests by utilizing "microorganisms that protect plants from pathogenic fungi" and "microorganisms that protect plants from harmful insects" that live in nature. It is expected to contribute greatly to ensuring food safety and security, as it has less impact on the environment.

There are many examples of using a filamentous fungus as a technique relating to a microbial control agent. For example, rice containing a filamentous fungus belonging to the genus Talaromyces having an antagonistic action against a pathogen that causes a disease during rice seedling raising, (Patent Document 1), which is effective for controlling various plant diseases and which does not show pathogenicity to main crops, and which enables control of various crop diseases by a kind of microorganism. A plant disease control agent containing Fusarium oxysporum NPF-9901 strain (FERM P-20469) (Patent Document 2), spores of Pythium oligandrum, and a calcium salt as a control effect enhancer, and a method for producing the same. In addition, a plant disease control method (Patent Document 3) using the plant disease control agent is known.

As a technology for microbial control agents using bacteria, a method for controlling rice seedling bacterial bacterial blight, which is planted in soil after immersing rice paddy in a suspension containing pathogenic deficient Erwinia carotovora bacteria (Patent Document 4), a controlling agent for the deadly disease of rice seedlings (Patent Document 5), which is characterized by containing Pseudomonas sp. CAB-02 as an active ingredient, is known.

Also, artificially infecting plants with bacteria having the ability to impart resistance to disease caused by pathogenic filamentous fungi, pathogenic bacteria or pathogenic viruses to host plants in symbiosis with the plant, pathogenic filamentous fungi in plants, In a method for controlling a disease caused by a pathogenic bacterium or a pathogenic virus, it is disclosed that a novel bacterium of the genus Herbaspirillum (accession number NITE BP-193) exhibits a disease resistance-inducing effect on rice blast (Patent Document 6). ).

Furthermore, the inventors of the present application have proposed that the Herbaspirillum sp. 022S4-11 strain (Patent Documents 7 and 8), Pantoea ananatis culture liquid (Patent Document 9), and bacterial cell suspension. It has been found that the above-mentioned and the supernatant liquid show an excellent bacterial disease control effect against rice bacterial wilt disease (seedling rot), rice seedling wilt disease, and rice brown stripe disease.

JP, 2007-31294, A JP, 2007-82499, A JP, 2010-143876, A JP-A-6-87716 JP-A-9-124427 International Publication No. 2007/100162 JP 2012-92093 A JP, 2005-028080, A JP, 2005-59090, A

Bacterial diseases that occur during the seedling raising period of rice plants include rice bacterial wilt disease (seedling rot), rice seedling bacterial wilt disease, and rice brown stripe disease. Rice seedling bacterial disease (seedling rot), rice seedling wilting bacterial disease and rice brown stripe disease, when sowing seeds carrying pathogenic bacteria, also infect healthy seedlings and cause seedling rot symptoms at the seedling raising stage. It becomes impossible to plant healthy seedlings in the paddy field, and eventually yield and quality are reduced. However, it is difficult to predict the occurrence of rice bacterial blight (seedling rot), rice seedling bacterial blight, and rice brown stripe disease, and once they occur, the rapid growth of bacteria cannot be suppressed even with the use of pesticides. It is also known that the sufficient effect cannot be obtained.

These pathogenic bacteria are thermophilic, and the occurrence of damage has a history of increasing with the spread of heated seedlings since the 1960s. Has become.

Until now, the control of these diseases at the time of raising seedlings has been centered on seed disinfection using chemically-synthesized pesticides, but in recent years, the treatment of waste liquid after disinfection has become a problem. Is becoming

Therefore, an object of the present invention is to provide a technique relating to a microbial pesticide which is effective against a bacterial disease occurring in the seedling raising stage of a grass family plant and has a low environmental load.

As a result of diligent studies by the present inventors, it was found that a new genus Herbaspirillum bacterium effectively controls a bacterial disease occurring in the seedling raising stage of a grass family plant. The present invention has been made based on such findings, and provides a harbor Spiritus ram sp (Herbaspirillum sp.) S4 strain (consignment number NITE P -2203).

The present invention also harbor Spiritus ram sp (Herbaspirillum sp.) Broth s4 strain (consignment number NITE P -2203), is selected from the group consisting of the supernatant of the bacterial cell suspension or the culture broth The present invention provides a bacterial disease controlling agent for controlling bacterial diseases of gramineous plants, which comprises at least one kind.

The present invention also provides a method for controlling bacterial diseases of gramineous plants, which comprises a control treatment step of attaching seeds of gramineous plants to the agent for controlling bacterial diseases.

The present invention also harbor Spiritus ram sp (Herbaspirillum sp.) Broth s4 strain (consignment number NITE P -2,203), is selected from the group consisting of the supernatant of the bacterial cell suspension or the culture broth The present invention also provides seeds comprising at least one kind of seeds, which is coated with a bacterial disease control agent for controlling bacterial diseases of gramineous plants.

Harbor Spiritus ram sp of the present invention (Herbaspirillum sp.) S4 strain according to (entrusted number NITE P -2,203) and bacterial disease control agent and control method using the strain, bacterial in seedling stage grasses Diseases such as rice blight bacterial disease (seedling rot), rice seedling blight bacterial disease and rice brown stripe disease, which are diseases, are effectively suppressed, and an extremely high control effect is obtained.

Further, according to the seed of the present invention, since the seeds of the rice plant are coated with a bacterial disease control agent effective for controlling the bacterial disease of the grass plant, the rice bacterial blight (seedling rot), It has resistance to bacterial seedling wilt disease and rice brown stripe disease, and can suppress the onset of bacterial diseases during seedling raising. Further, after coating with the bacterial disease controlling agent, the effect continues, and thus the seeds can be distributed as they are.

Herbaspirillum genus bacteria are bacteria isolated from healthy Gramineae plants, Solanaceae plants, Cucurbitaceae plants, Brassicaceae plants, Rosaceae plants, soil and water. It can contribute to stable production of gramineous plants in environment-friendly agriculture that is environmentally friendly.

1. Bacterial diseases controlling agent of bacterial diseases of gramineous plants embodiment of control agent present invention of bacterial diseases of gramineous plants, harbor Spiritus ram sp (Herbaspirillum sp.) S4 strain (consignment number NITE P -2 203 ), the culture solution, the bacterial cell suspension, or the supernatant of the culture solution.

The bacterial disease control agent of the present embodiment exhibits an effect of controlling bacterial diseases of gramineous plants, specifically, Burkholderia glumae, which is a causative bacterium of rice bacterial blight fungus, Burkholderia plantarii, which is the causative bacterium of bacterial wilt disease of rice, and Acidovorax avenae, which is the causative bacterium of brown stripe of rice, exert a disease suppressive effect.

Incidentally, the bacterial disease control agent of the present embodiment is not directly antagonistic capacity against pathogenic bacteria, harbor Spiritus ram sp (Herbaspirillum sp.) S4 strain broth (consignment number NITE P -2 203), bacteria It is presumed that some substance contained in the body suspension or the supernatant of the culture medium acts to suppress the disease.

Harbor Spiritus ram sp (Herbaspirillum sp.) If s4 strain broth (consignment number NITE P -2 203) used as an active ingredient of bacterial disease control agent, harbor Spiritus ram sp (Herbaspirillum sp.) S4 strain the (entrusted number NITE P -2,203) can be used directly or diluted with, or concentrated for a predetermined time cultured broth in a liquid medium for the bacteria (such as potato semisynthetic agar, etc.).

Harbor Spiritus ram sp (Herbaspirillum sp.) If s4 strain Cells of (consignment number NITE P -2 203) used as an active ingredient of bacterial disease control agent, harbor Spiritus ram sp (Herbaspirillum sp.) S4 strain separated (entrusted number NITE P -2 203) the culture medium using centrifugation for culture a predetermined time cultured in a liquid medium for the bacteria (such as potato semisynthetic agar medium, etc.) into cells and supernatant Then, a microbial cell suspension obtained by suspending the obtained microbial cell in a solvent such as water can be prepared and used. In this case, the bacterial cell concentration of the bacterial cell suspension is preferably at least 10 ⁶ cfu/ml or more, more preferably 10 ⁷ cfu/ml or more, and 10 ⁸ cfu/ml or more. More preferable.

In the present embodiment, it includes crushed the cell suspension harbor Spiritus ram sp (Herbaspirillum sp.) S4 strain (consignment number NITE P -2203). When using harbor Spiritus ram sp (Herbaspirillum sp.) S4 strain crushed (consignment number NITE P -2,203) as an active ingredient of bacterial diseases control agent, and using a homogenizer or the like and disrupted, It can be used as a suspension of disrupted cells. The bacterial cell concentration before crushing is based on the case of preparing the above-mentioned bacterial cell suspension.

Harbor Spiritus ram sp (Herbaspirillum sp.) If s4 strain supernatants of the culture solution of (consignment number NITE P -2 203) used as an active ingredient of bacterial disease control agent, Herba Spiritus ram (Herbaspirillum) bacterium Was cultivated in a liquid medium for bacteria (for example, potato semi-synthetic agar) for a certain period of time, and the culture solution was separated into cells and supernatant using centrifugation, membrane separation, filtration separation, etc. The resulting supernatant can be used as it is, diluted or concentrated.

(. Herbaspirillum sp) harbor Spiritus ram sp s4 strain (consignment number NITE P -2 203) is a Gram-negative bacteria isolated from grasses, in the present embodiment, at least a healthy rice (leaf sheaths, cultivar: Koshihikari), sugar cane pseudo-spot disease spots, eutrophic lake water, soil sediment, ogi (leaf), distilled water, fresh water, and at least one type isolated from rice (root) can also be used. .. Harbor Spiritus ram sp (Herbaspirillum sp.) S4 strain (consignment number NITE P -2 203) is nitrogen-fixing ability also has, preferred because it can be utilized such properties in the cultivation of gramineous plants.

The present inventors have harbor Spiritus ram sp (Herbaspirillum sp.) S4 strain was subjected to comparison with (entrusted number NITE P -2 203) and Herba Spiritus ram (Herbaspirillum) genus known species, the previously known strains because it can be clearly distinguished, which was identified as a new strain has been deposited with the National Institute of Advanced industrial Science and technology Patent organism Depositary Center (NITE-IPOD) (consignment number NITE P -2203). Harbor Spiritus ram sp (Herbaspirillum sp.) Features s4 strain (consignment number NITE P -2203) is as follows.

(1) 16S rDNA of Herbaspirillum sp. strain (Herbaspirillum sp.) (accession number NITE P-2203) which was subjected to sequence analysis based on the phylogenetic tree based on the nucleotide sequence of 16S rDNA and homologous species nucleotide sequence (about 700b p) and Herba Spiritus ram (Herbaspirillum) of known species and species name undetermined genera harbor Spiritus ram that is registered in the database (GenBank + EMBL + DDBJ + PDB ) , which is published on the Internet (Herbaspirillum) spp. (B501: Isolation source wild rice, BA17: Isolation source banana) Based on the nucleotide sequence of 16S rDNA, CLUSTAL W (1.83) was used for alignment, and a phylogenetic tree was created by NJ method. .. The out group used was R. solanacearum of the closely related genus Ralstonia.

As a result, harbor Spyri lamb sp s4 share (consignment number NITE P -2203), the harbor Spyri Lam Le Bliss Balbi albicans (Herbaspirillum sp.): Was (Herbaspirillum rubrisubalbicans sugar cane pseudo red streaks fungus) and the closely related .. In addition, the previous database (GenBank + EMBL + DDBJ + PDB ) BLASTN using (2.2.13) harbor Spyri lamb sp in search (Herbaspirillum sp.) S4 shares (consignment number NITE P -2203) and Herba Spyri lamb・The homology with Lubris barbicans (Herbaspirillum rubrisubalbicans) was 98% or more.

(2) Bacteriological properties (API20NE)
Using API20NE a bacterial test kit result of studying the bacteriological properties, harbor Spiritus ram sp (Herbaspirillum sp.) S4 strain known to be consistent with the bacteriological properties of the (fiduciary number NITE P -2,203) bacteria There was no seed.

(3) Taxonomic position (identification)
Harbor Spiritus ram sp (Herbaspirillum sp.) S4 strain (consignment number NITE P -2 203) is Herba phylogenetic analysis was based on the nucleotide sequence of 16S rDNA from its homology Spiritus ram le Bliss barbituric albicans (Herbaspirillum Rubrisubalbicans ), but it was suggested that they are independent of each other.

Furthermore, expression harbor Spiritus ram sp in bacteriological properties are transformed (Herbaspirillum sp.) S4 strain (consignment number NITE P -2,203) is stored in the MAFF Genebank harbor Spiritus Lamb Ruburisu None of the 29 strains of Barbicans (Herbaspirillum rubrisubalbicans) matched their properties.

Harbor Spiritus ram sp (Herbaspirillum sp.) S4 strain (consignment number NITE P -2203) and harbor Spiritus ram le Bliss barbituric albicans (Herbaspirillum rubrisubalbicans) showed a very high homology in the nucleotide sequence of 16S rDNA , be different bacteriological properties in phenotypically, further harbor Spiritus ram sp (Herbaspirillum sp.) s4 strain (consignment number NITE P -2203) from the lack pathogenicity in sugar cane, harbor Spiritus ram sp (Herbaspirillum sp.) s4 strain (entrusted number NITE P -2203) determines that the herbalist Spyri lamb (Herbaspirillum) genus and species of, was identified as the harbor Spyri lamb sp (Herbaspirillum sp.).

2. Method for Controlling Bacterial Diseases of Gramineous Plants The method for controlling a bacterial disease of Gramineae plants of the present embodiment has a control treatment step of attaching seeds of Gramineae plants to the above-mentioned bacterial disease control agent.

There is no particular limitation on the method of attaching the seeds of the grass family to the bacterial disease control agent, but the control treatment step is, from the viewpoint of more reliably attaching the bacterial disease control agent to the seeds, the bacterial disease control agent of the grass family plant. The step of soaking the seeds is preferable.

The control treatment step is preferably carried out for at least 12 hours or more, and may be carried out at any time before sowing, before soaking, during soaking, and after soaking (that is, during germination), as long as the seeds do not germinate.

Here, "soaking" is a treatment step before germination, and refers to a step of allowing seeds to absorb water so as to germinate all at once. Immersion is performed, for example, by immersing in warm water at about 15 to 30° C. for about 3 to 4 days. Usually, seeds with low solidity are removed or sterilized in advance by water selection or salt water selection (putting seeds in water or salt water for selection) and soaking. "Emergence" refers to an artificial treatment that promotes the initiation of bud development, the initiation of dormant bud development, and uniform germination. The germination is carried out, for example, by immersing in warm water of about 20 to 35° C. for about 12 to 24 hours.

Herba Spyri Ram sp (Herbaspirillum sp.) S4 shares (consignment number NITE P -2203) the binding properties of the seeds of grasses are inherently superior. Therefore, it is not necessary to use an additive such as a binder when coating the seeds of Gramineae with these bacteria of the genus Herbaspirillum.

It should be noted that although a certain effect can be obtained even if the control treatment is carried out after the seeds of the Gramineae plant have sprouted, the seeds carrying the rice seedling blight fungus, the rice blight fungus or the rice brown stripe fungus are healthy. In many cases, it is impossible to germinate, and the controlling effect is lower than that in the case where the controlling treatment is carried out before emergence.

3. Seeds Seeds of this embodiment, harbor Spiritus ram sp (Herbaspirillum sp.) Cell or disrupted product thereof s4 strain (consignment number NITE P -2203) or harbor Spiritus ram sp (Herbaspirillum sp.) S4 strain ( wherein the culture solution or a supernatant of the consignment number NITE P -2203), in which the rice plant bacterial effective bacterial disease control agent for controlling diseases, was coated seeds of gramineous plants.

The other composition of the bacterial disease controlling agent is similar to the composition of the bacterial disease controlling agent described above. The method for coating the seeds of the Gramineae plant with the bacterial disease control agent is based on the method for controlling the bacterial disease of the Gramineae plant described above.

The Gramineae plant applicable to the present embodiment refers to a plant belonging to the Gramineae family in plant taxonomy, and examples thereof include rice, wheat, barley, corn, millet, and millet. Among the Gramineae plants, rice (Oryza sativa) is preferable.

According to the seed of the present embodiment, since the seeds of the Gramineae plant are coated with a bacterial disease control agent effective for controlling the bacterial disease of the Gramineae plant, in the process of germination and germination of the seed, the rice seedlings are dead. It has resistance to bacterial diseases (seedling rot), bacterial seedling wilting disease or rice brown stripe disease, and can suppress the onset of these bacterial diseases during seedling raising.

In addition, after the seeds are coated with the bacterial disease controlling agent, the effect continues as long as the bacterial disease controlling agent is coated, so that, for example, seeds coated with the bacterial disease controlling agent can be distributed as they are.

1. Herbaspirillum sp. s4 Strain Strain Strain Suppressive Effect on Rice Blight Bacterial Disease (1) Seedling Conditions Rice seedlings were raised under the following conditions. For seeding, put seedling cultivation soil (Iseki cultivation soil) in a plastic case (35 mm × 110 mm × 110 mm) with 5 holes with a diameter of about 1 mm at the bottom for drainage, and 15 g of treated seed paddy is evenly seeded and lightly covered with soil. did. In addition, after sowing, it was managed in a glass greenhouse.

(2) Method for producing contaminated paddy Seed pad is soaked in a suspension (about 10 ⁸ cfu/ml) of Burkholderia glumae MAFF301441 strain, which is a bacterial fungus of rice blight, under vacuum reduced pressure for about 10 minutes After inoculating the seed paddy with pathogenic bacteria, the drained seed paddy was spread on a lab towel or the like and air-dried at room temperature overnight.

(3) Each processing solution prepared and processing method harbor Spiritus ram sp of (Herbaspirillum sp.) S4 strain (consignment number NITE P -2 203) (hereinafter referred to simply as "s4 share") a vibration in PPG medium Culture solution (hereinafter referred to as "culture solution") that has been subjected to culturing (25°C, 2 days, 100 rpm), and culture supernatant solution obtained by centrifugation of the culture solution (hereinafter referred to as "supernatant solution") )) and microbial cells obtained by centrifugation with the same amount of sterilized water as the supernatant removed by centrifugation to suspend the microbial cell suspension (hereinafter referred to as “microbial cell suspension”). Was prepared and used for the test.

After soaking seed rice containing contaminated paddy of rice blast fungus, treatment is carried out by immersing the seed rice after soaking in each of the culture solution, supernatant liquid and cell suspension for 16 hours at 32°C. It was After that, the seed paddy was subjected to budding treatment, and the disease suppressive effect against rice bacterial blight was investigated. Distilled water was used as an untreated section according to each usage method.

(4) Survey method The disease severity was investigated for all seedlings in each ward, and an index was given for each severity, and the disease severity was calculated by the diseased seedling rate and the following formula. The indices were: dead seedlings: 5, diseased seedlings other than death (whitening/dwarfing/abnormal extraction): 3, healthy seedlings: 0.
Disease severity = {Σ (number of seedlings by disease severity x index) / (5 x number of survey seedlings)} x 100
Control value = (1-the degree of disease in the treated area/the degree of disease in the untreated area) x 100

(5) Results Table 2 shows the examination results of the disease suppressive effect of the s4 strain on the rice bacterial blight caused by the soaking treatment. As shown in Table 2, when the seed paddy was treated with the culture solution of the s4 strain, the bacterial cell suspension and the supernatant, the control value was high, and the bacterial disease control agent of the present invention showed that the rice blast bacterium It was found to be effective against the disease. On the other hand, in the non-treated area (distilled water treatment), the bacterial bacterial blight of rice occurred frequently and the control value could not be evaluated.

2. Herbaspirillum sp. s4 Strain Strain Suppressive Effect on Seedling Bacterial Disease of Rice (1) Seedling Conditions Rice seedlings were raised under the same conditions as described in 1 (1) above. ..

(2) Method for producing contaminated paddy The contaminated paddy was produced by the same procedure as in 1 (2) above, except that the pathogenic bacterium was Burkholderia plantarii MAFF301723 strain, which is a bacterial pathogen of rice seedling blight.

(3) Preparation and treatment method of each treatment liquid By the same operation as in 1 (3) above, a culture liquid, a supernatant liquid and a bacterial cell suspension of the s4 strain were prepared and subjected to the test.

(4) Investigation method The diseased seedling rate and disease severity were calculated by the same calculation method as in 1 (4) above.

(5) Results Table 3 shows the examination results of the disease suppressing effect of the s4 strain on the seedling wilting bacterial disease of rice in the treatment during soaking. As shown in Table 3, when the seed paddy was treated with the culture solution of the s4 strain, the bacterial cell suspension and the supernatant, the control value was high, and the bacterial disease control agent of the present invention showed that the rice seedlings were dead. It has been found to be effective against bacterial diseases. On the other hand, in the untreated area (distilled water treatment), bacterial seedling wilting disease frequently occurred, and the control value could not be evaluated.

3. Herbaspirillum sp. S4 Strain Strain Suppressing Effect on Brown Streak of Rice (1) Seedling Conditions Rice seedlings were raised under the same conditions as described in 1 (1) above.

(2) Method for producing contaminated paddy The contaminated paddy was produced by the same procedure as in 1 (2) above, except that the pathogenic bacterium was Acidovorax avenae MAFF301752 strain, which is a rice stripe fungus.

(3) Preparation and treatment method of each treatment liquid By the same operation as in 1 (3) above, a culture liquid, a supernatant liquid and a bacterial cell suspension of the s4 strain were prepared and subjected to the test.

(4) Investigation method The diseased seedling rate and disease severity were calculated by the same calculation method as in 1 (4) above.

(5) Results Table 4 shows the examination results of the disease suppressive effect of the s4 strain on the rice stripe disease in the soaking process. As shown in Table 4, when the seed rice was treated with the culture solution of the s4 strain and the bacterial cell suspension, both showed high control value, and the bacterial disease control agent of the present invention is effective against rice stripe disease. It turned out to be. On the other hand, in the non-treated area (treated with distilled water), brown stripes of rice frequently occurred, and the control value could not be evaluated.

Claims (8)

Herba Spyri Ram sp (Herbaspirillum sp.) S4 shares (consignment number NITE P -2203). Harbor Spiritus ram sp (Herbaspirillum sp.) Broth s4 strain (consignment number NITE P -2203), comprises at least one member selected from the group consisting of the supernatant of the bacterial cell suspension or the culture broth , bacterial diseases controlling agent for controlling a bacterial disease of rice plant. The bacterial disease control according to claim 2, wherein the bacterial disease is at least one selected from the group consisting of rice seedling bacterial disease (seedling rot), rice seedling wilt disease, and rice brown stripe disease. Agent. A method for controlling bacterial diseases of grasses, which comprises a control treatment step of attaching seeds of grasses to the agent for controlling bacterial diseases according to claim 2 or 3. The method for controlling bacterial diseases of gramineous plants according to claim 4, wherein the control treatment step is a step of immersing the seeds in the bacterial disease control agent. The method for controlling bacterial diseases of gramineous plants according to claim 4 or 5, wherein the controlling step is carried out for at least 12 hours or more. Harbor Spiritus ram sp (Herbaspirillum sp.) Broth s4 strain (consignment number NITE P -2 203), comprises at least one member selected from the group consisting of the supernatant of the bacterial cell suspension or the culture broth A seed in which a seed of a Gramineae plant is coated with a bacterial disease control agent for controlling a bacterial disease of a Gramineae plant. The seed according to claim 7, wherein the gramineous plant is rice (Oryza sativa).