JP2012092093A - Controlling agent and controlling method for bacterial disease of gramineous plant, and seed coated with the controlling agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technology related to microbial pesticide that is effective against a bacterial disease occurring in a seedling growing season of a gramineous plant and scarcely puts a load on the environment.SOLUTION: This bacterial disease controlling agent includes herbaspirillum-group bacteria, their debris, culture solution of the herbaspirillum-group bacteria, its supernatant solution, and is effective in controlling the bacterial disease of the gramineous plant. This controlling method of the bacterial disease of the gramineous plant has a controlling process of bonding it to the bacterial disease controlling agent. This seed of the gramineous plant is coated with the bacterial disease controlling agent.

Description

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

  In modern agriculture, pest control technology centered on so-called chemical pesticides has been developed in order to secure food efficiently. However, as a result of excessive use of chemical pesticides for many years, problems such as ecosystem disruption, food safety due to residual pesticides, and health hazards for farmers using chemical pesticides have been highlighted. Therefore, there is a demand for conversion to pesticides with low toxicity and persistence, and from there, efforts to reduce pesticides and pesticides are being demanded.

  In such a trend, attention has been focused on pest control technologies (for example, microbial control agents) that are suitable for environmental conservation agriculture that takes into consideration the reduction of the environmental burden caused by the use of agricultural chemicals. The term “microbe control agent” refers to a preparation that protects crops from damage caused by pests by utilizing “microorganisms that protect plants from pathogenic bacteria” and “microorganisms that protect plants from pests” that exist in nature. It is expected to contribute greatly to ensuring food safety and security with less impact on the environment.

  There are many examples of techniques related to microbial control agents that use filamentous fungi. For example, rice containing a filamentous fungus belonging to the genus Talaromyces that has an antagonistic action against pathogenic bacteria that cause diseases during the seedling raising period of rice. For controlling diseases that occur during the seedling raising period (Patent Document 1), Fusarium that is effective in controlling various plant diseases and that does not show pathogenicity in major crops, and that can control various crop diseases with a single microorganism Plant disease control agent comprising Fusarium oxysporum NPF-9901 strain (FERM P-20469) (Patent Document 2), Psium origandrum spore, and calcium salt as a control effect enhancing substance, and method for producing the same In addition, a plant disease control method (Patent Document 3) using the same plant disease control agent is known.

  As a technique related to a microbial control agent using bacteria, a method for controlling a rice seedling bacterial disease that is planted in soil after immersing rice bran in a suspension containing Erwinia carotobola bacteria lacking pathogenicity (Patent Document 4), Pseudomonas sp. (Pseudomonas sp.) CAB-02 is contained as an active ingredient, and a rice seedling disease control agent (Patent Document 5) is known.

  In addition, the plant is artificially infected with a bacterium that has the ability to symbiotically exist in the plant body and imparts resistance to diseases caused by pathogenic filamentous fungi, pathogenic bacteria or pathogenic viruses to the host plant, In a method for controlling diseases caused by pathogenic bacteria or pathogenic viruses, it is disclosed that a novel bacterium belonging to the genus Herbaspirillum (Accession No. NITE BP-193) exhibits a disease resistance-inducing effect against rice blast (Patent Document 6). ).

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

  Bacterial diseases that occur during the seedling stage of gramineous plants include rice seedling blight, rice foliage (seed rot) and rice brown streak. Rice seedling bacterial disease, rice blast bacterial disease and rice brown streak disease can infect healthy seedlings when seeds bearing pathogenic bacteria are sown, causing seed rot symptoms during the seedling season, Cannot be planted, resulting in a decrease in yield and quality. However, it is difficult to predict the occurrence of rice seedling blight disease, rice blast blight disease and rice brown streak disease, and once they occur, the rapid growth of the bacteria cannot be suppressed even with the use of pesticides. It is also known that it cannot be obtained.

  These pathogenic bacteria are hyperthermophilic, and the occurrence of damage has increased with the spread of warming seedlings since the 1960s, and is now an important disease at the seedling stage, along with rice fungal disease, which is a filamentous fungal disease. It has become.

  Until now, the control of these diseases at the time of seedling has been centered on seed disinfection using chemically synthesized pesticides, but in recent years it has become important to consider the environment, such as waste liquid treatment after disinfection. It has become.

  Accordingly, an object of the present invention is to provide a technique relating to a microbial pesticide that is effective for bacterial diseases occurring in the seedling growing period of gramineous plants and has a low environmental load.

  As a result of intensive studies by the present inventors, it has been found that bacteria belonging to the genus Herbaspirillum can effectively control bacterial diseases that occur during the seedling stage of gramineous plants. The present invention has been made based on such findings, and provides a bacterial disease control agent that contains a culture solution of Herbaspirillum bacteria or a supernatant thereof, and is effective in controlling bacterial diseases of gramineous plants. To do.

  The present invention also provides a bacterial disease control agent that contains a bacterium belonging to the genus Herbaspirillum or a crushed product thereof and is effective in controlling bacterial diseases of gramineous plants.

  Moreover, this invention provides the control method of the bacterial disease of the Gramineae plant which has the control process which attaches the seed of Gramineae plant to the said bacterial disease control agent.

  The present invention also provides Herbaspirillum sp. 022S4-11 strain (reception number FERM AP-22001).

  In addition, the present invention includes a Herbaspirillum genus bacterium or a crushed product thereof or a culture solution of Herbaspirillum genus bacterium or a supernatant thereof, which is effective for controlling bacterial diseases of gramineous plants. The present invention provides seeds obtained by coating bacterial seeds with a bacterial disease control agent.

  According to the bacterial disease control agent and the bacterial disease control method of the present invention, pathogenic diseases such as rice seedling bacterial disease and rice wilt bacterial disease (seedling rot), which are bacterial diseases in the seedling growing season of gramineous plants, are caused. It is effectively suppressed and an extremely high control effect is obtained.

  In addition, according to the seed of the present embodiment, since the rice plant seed is coated with a bacterial disease control agent effective in controlling bacterial diseases of the grass family, It has resistance to bacterial diseases and can suppress the onset of bacterial diseases during seedling raising. In addition, after coating with a bacterial disease control agent, the effect persists thereafter, so that it can be distributed as seeds.

  Herbaspirillum bacteria are isolated from healthy gramineous plants, solanaceous plants, cucurbitaceae plants, cruciferous plants, rosetic plants, soil and water, without polluting the environment, It can contribute to the stable production of gramineous plants in environmentally friendly agriculture.

It is a figure which shows the process process of this experiment (The process at the time of germination, 022S4-11 strain | stump | stock, a rice blast blight disease). It is a figure which shows the process process of this experiment (The process at the time of soaking, 022S4-11 strain | stump | stock, a rice blast blight disease). It is a figure which shows the growth condition of the seedling about two weeks after sowing the seed pod treated with the cell suspension of the 022S4-11 strain. It is a figure showing the growth of seedlings about 2 weeks after sowing seed pods treated with the culture solution of 022S4-11 strain. It is a figure which shows the process process of this experiment (The process at the time of germination, 022S4-11 strain | stump | stock, a rice seedling blight bacterial disease). FIG. 10 is a diagram showing the growth of seedlings about 2 weeks after seeding seed pods treated with the supernatant of 022S4-11 strain. It is a figure which shows the growth condition of the seedling about two weeks after sowing the seed pod treated with the cell suspension of the 022S4-11 strain. It is a figure showing the growth of seedlings about 2 weeks after sowing seed pods treated with the culture solution of 022S4-11 strain. It is a figure which shows the process process of this experiment (The process at the time of soaking, 022S4-11 strain | stump | stock, a rice seedling blight bacterial disease). FIG. 10 is a diagram showing the growth of seedlings about 2 weeks after seeding seed pods treated with the supernatant of 022S4-11 strain. It is a figure which shows the growth condition of the seedling about two weeks after sowing the seed pod treated with the cell suspension of the 022S4-11 strain. It is a figure showing the growth of seedlings about 2 weeks after sowing seed pods treated with the culture solution of 022S4-11 strain. It is a figure which shows the process of this experiment (The process at the time of germination, 022S4-11 strain | stump | stock, rice brown stripe disease). It is a figure showing the growth of seedlings about 2 weeks after sowing seed pods treated with the culture solution of 022S4-11 strain. It is a figure which shows the growth condition of the seedling about two weeks after sowing the seed pod treated with the cell suspension of the 022S4-11 strain. FIG. 10 is a diagram showing the growth of seedlings about 2 weeks after seeding seed pods treated with the supernatant of 022S4-11 strain. It is a figure which shows the process process of this experiment (The process at the time of soaking, 022S4-11 strain | stump | stock, rice brown stripe disease). It is a figure showing the growth of seedlings about 2 weeks after sowing seed pods treated with the culture solution of 022S4-11 strain. It is a figure which shows the growth condition of the seedling about two weeks after sowing the seed pod treated with the cell suspension of the 022S4-11 strain. FIG. 10 is a diagram showing the growth of seedlings about 2 weeks after seeding seed pods treated with the supernatant of 022S4-11 strain. It is a figure which shows the process process of this experiment (The process at the time of germination, MAFF311406 strain | stump | stock, a rice blast blight disease). It is a figure which shows the growth condition of the seedling about two weeks after sowing the seed pod treated with the culture solution of MAFF311406 strain. It is a figure which shows the process process of this experiment (The process at the time of soaking, MAFF311406 strain | stump | stock, a rice wilt bacterial disease). It is a figure which shows the growth condition of the seedling about two weeks after sowing the seed pod treated with the culture solution of MAFF311406 strain. It is a figure which shows the growth condition of the seedling about two weeks after sowing the seed pod treated with the supernatant liquid of MAFF311406 strain. It is a figure which shows the growth condition of the seedling about 2 weeks after sowing the seed pod treated with the bacterial cell suspension of MAFF311406 strain. It is a figure which shows the process process of this experiment (The process at the time of germination, MAFF311406 strain | stump | stock, a rice seedling blight bacterial disease). It is a figure which shows the growth condition of the seedling about two weeks after sowing the seed pod treated with the processing solution of MAFF311406 strain. It is a figure which shows the process of this experiment (The process at the time of soaking, MAFF311406 strain, rice brown stripe disease). It is a figure which shows the growth condition of the seedling about two weeks after sowing the seed pod treated with the processing solution of MAFF311406 strain. It is a figure which shows the control effect with respect to a rice blast blight disease by the culture solution process of H. putei strain | stump | stock. It is a figure which shows the control effect with respect to the rice blast bacterial disease by the supernatant liquid process of H. putei strain | stump | stock. It is a figure which shows the control effect with respect to the rice blast blight disease by the cell suspension process of H. putei stock | strain. It is a figure which shows the growth promotion effect with respect to the rice seedling of 022S4-11 strain. It is a figure which shows the phylogenetic tree based on the base sequence of 16S rDNA.

1. Bacterial disease control agent of gramineous plant The bacterial disease control agent of bacterial disease of gramineous plant of the embodiment of the present invention is Herbaspirillum spp., Herbaspirillum spp. And a supernatant of a culture solution of Herbaspirillum bacteria or a culture solution of Herbaspirillum bacteria.

The bacterial disease control agent of the present embodiment exerts an effect on the control of bacterial diseases of gramineous plants, and specifically, Burkholderia plantarii which is a causative fungus of the seedling bacterial disease. And Burkholderia gourmet (Burkholderia gourmet)
glumae) against disease. In addition, the bacterial disease control agent of this embodiment does not have direct antagonism against pathogenic bacteria, and is a Herbaspirillum genus bacterium, Herbaspirillum genus crushed material, Herbaspirillum genus Herbaspirillum (Herbaspirillum) ) It is presumed that some substance contained in the culture solution of the genus bacteria or the supernatant of the culture solution of the genus bacteria of Herbaspirillum acts to suppress disease.

When using bacteria from the genus Herbaspirillum as an active ingredient in a bacterial disease control agent, use the Herbaspirillum bacterium in a liquid medium for bacteria (such as a potato semi-synthetic agar medium). For culture broth that has been cultured for a long time, the culture broth is separated into cells and supernatant using centrifugation, etc., and a cell suspension is prepared by suspending the obtained cells in a solvent such as water. can do. In this case, the cell concentration of the cell suspension is preferably at least 10 ⁶ cfu / ml or more, more preferably 10 ⁷ cfu / ml or more, and preferably 10 ⁸ cfu / ml or more. Further preferred.

  When using disrupted Herbaspirillum bacteria as an active ingredient in a bacterial disease control agent, disrupt the cells using a homogenizer, etc., and use them as a suspension of disrupted cells. it can. The cell concentration before crushing is in accordance with the case of preparing the above-described cell suspension.

  When using a culture solution of Herbaspirillum bacteria as an active ingredient of a bacterial disease control agent, use the Herbaspirillum bacteria in a liquid medium for bacteria (for example, potato semi-synthetic agar medium) The culture solution which has been cultured for a long time can be used as it is or after being diluted or concentrated.

  When using the supernatant of the culture solution of the genus Herbaspirillum as an active ingredient of a bacterial disease control agent, use the Herbaspirillum bacterium in a liquid medium for bacteria (eg, potato semi-synthetic agar medium) Etc.), the culture solution is separated into cells and supernatant using centrifugation, membrane separation, filtration separation, etc., and the resulting supernatant is diluted or concentrated as it is. Can be used.

  The genus Herbaspirillum is a Gram-negative bacterium isolated from the grass family, and in this embodiment, Herbaspirillum is isolated from at least healthy rice (leaf sheath, variety: Koshihikari). sp.) or Herbaspirillum rubrisubalbicans isolated from sugarcane pseudo-Red stripe disease, Herbaspirillum autotrophicum isolated from eutrophic lake water, soil deposition Herbaspirillum chlorophenolicum isolated from food, Herbaspirillum frisingense isolated from ogi (leaves), Herbaspirillum huttiense isolated from distilled water Harbus, isolated from freshwater It is preferable to use at least one selected from Herbaspirillum putei and Herbaspirillum seropedicae isolated from rice (root). Among Herbaspirillum sp. (Herbaspirillum sp.), Herbaspirillum sp. 022S4-11 strain (reception number FERM AP-22001) has a nitrogen-fixing ability. Such cultivation is preferable because it can be used for cultivation.

  Herbaspirillum sp. 022S4-11 (accession number FERM AP-22001) is a bacterium isolated from the leaf sheath of healthy rice. When the present inventors compared the Herbaspirillum sp. 022S4-11 strain with a known species of the genus Herbaspirillum, it can be clearly distinguished from conventionally known strains. As a result, it was identified as a new strain and deposited at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary. The characteristics of Herbaspirillum sp. 022S4-11 are as follows.

(1) Phylogenetic tree based on 16S rDNA base sequence and homology with related species 16S rDNA base sequence (1,179 bp) of Herbaspirillum sp. 022S4-11 strain subjected to sequence analysis , AB259359) and the publicly available databases (GenBank + EMBL + DDBJ + PDB) of the genus Herbaspirillum and herb undetermined genus Herbaspirillum After alignment with CLUSTAL W (1.83) based on the base sequence of 16S rDNA of (B501: isolation source wild rice, BA17: isolation source banana), a phylogenetic tree was created by the NJ method. In addition, R.solanacearum of the genus Ralstonia (Ralstonia) (Ralstonia) was used for Out group.

As a result, the 022S4-11 strain was identified as Herbaspirillum Herbaspirillum
rubrisubalbicans: closely related to sugarcane pseudo-red streak fungus). In the BLASTN (2.2.13) search using the previous database (GenBank + EMBL + DDBJ + PDB), Herbaspirillum
sp.) 022S4-11 and Herbaspirillum rubris barbicans (Herbaspirillum)
rubrisubalbicans) was 99% homologous (see FIG. 35).

(2) Bacteriological properties (API20NE)
As a result of examining bacteriological properties using API20NE, a bacterial test kit, there was no known bacterial species consistent with the bacteriological properties of Herbaspirillum sp. 022S4-11 strain. Table 1 shows the bacteriological properties of Herbaspirillum sp. 022S4-11.

(3) Taxonomic position (identification)
Herbaspirillum sp. 022S4-11 is closely related to Herbaspirillum rubrisubalbicans due to its phylogenetic analysis based on the 16S rDNA base sequence and its homology. It was suggested that these are independent relationships.

  Furthermore, in terms of bacteriological properties as a phenotype, the Herbaspirillum sp. 022S4-11 strain is Herbaspirillum rubrisubalbicans 29 preserved in the Genebank of the Ministry of Agriculture, Forestry and Fisheries 29 There was no strain that matched the nature of the strain.

  Herbaspirillum sp. 022S4-11 and Herbaspirillum rubrisubalbicans showed very high homology in the 16S rDNA sequence, but bacteriology in phenotypes Herbaspirillum sp. 022S4-11 has no pathogenicity to sugarcane, so Herbaspirillum sp. 022S4-11 was replaced by Herbaspirillum sp. ) Was determined to be a new species of the genus and identified as Herbaspirillum sp.

2. Control Method for Bacterial Diseases of Gramineae Plants The control method for bacterial diseases of gramineous plants according to the present embodiment includes a control treatment step of attaching the seeds of the gramineous plants to the above-mentioned bacterial disease control agent.

  There is no particular limitation on the method for attaching the seeds of the gramineous plant to the bacterial disease control agent. However, the control treatment process is more effective in attaching the bacterial disease control agent to the seed from the viewpoint of adhering the bacterial disease control agent to the seeds more reliably. A step of immersing seeds is preferred.

  The control treatment step is preferably performed for at least 12 hours or more, and may be performed before soaking, during soaking, or after soaking (that is, during germination) as long as the seeds are not germinated.

  Here, “immersion seed” refers to a treatment step before germination, which is a step of allowing seeds to absorb moisture so that they germinate all at once. For example, the immersion is performed by immersing in warm water of about 15 to 30 ° C. for about 3 to 4 days. Usually, seeds with low solidity are removed in advance by water selection or salt water selection (sorting seeds in water or salt water) or sterilized after sterilization. “Sprouting” refers to an artificial treatment that promotes the formation of new shoots or the start of development of dormant shoots, or makes germination uniform. Germination is performed, for example, by immersing in warm water at about 20 to 35 ° C. for about 12 to 24 hours.

  In addition, although a certain effect is obtained even if the control treatment is performed after the seeds of the Gramineae plants emerge, the seeds carrying the rice seedling bacterial bacterium, the rice blast fungus or the rice brown streak are healthy. In many cases, germination cannot be germinated, and the control effect is reduced as compared with the case where the control treatment is performed before germination.

3. Seeds The seeds of this embodiment contain Herbaspirillum bacteria or their disruptions or Herbaspirillum bacteria culture solutions or supernatants thereof, for controlling bacterial diseases of gramineous plants. The seeds of gramineous plants are coated with an effective bacterial disease control agent.

  Other configurations of the bacterial disease control agent are in accordance with the above-described configurations of the bacterial disease control agent. In addition, the method of coating the seeds of the above gramineous plants with the bacterial disease control agent is in accordance with the above-described method for controlling bacterial diseases of gramineous plants.

  Herbaspirillum sp., A bacterium that can be used as a genus of Herbaspirillum genus, is a bacterium isolated from healthy grasses, Herbusspirillum rubrisbarbi Kangsu (Herbaspirillum rubrisubalbicans) is a bacterium isolated from the lesion of the sugarcane pseudo-red-head disease, and Herbaspirillum autotrophicum is a bacterium isolated from eutrophic lake water. Herbaspirillum chlorophenolicum is a bacterium isolated from soil sediments, and Herbaspirillum frisingense is a bacterium isolated from ogi (leaves).・ Hatchense (Herbaspirillum huttiense) is a bacterium isolated from distilled water. Herbaspirillum putei is a bacterium isolated from fresh water, and Herbaspirillum seropedicae is a bacterium isolated from rice (root). All of these Herbaspirillum bacteria are inherently excellent in binding to the seeds of Gramineae plants. Therefore, it is not necessary to use an additive such as a binder when coating these Herbaspirillum bacteria on the seeds of gramineous plants.

  In particular, the Herbaspirillum sp. 022S4-11 strain (reception number FERM AP-22001) is preferable because it is a bacterium isolated from a healthy rice leaf sheath. Herbaspirillum sp. 022S4-11 (reception number FERM AP-22001) has been confirmed to have nitrogen-fixing ability by the acetylene reduction method and promotes the growth of rice seedlings. There is also an effect.

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

  According to the seeds of this embodiment, since the seeds of gramineous plants are coated with a bacterial disease control agent effective for controlling bacterial diseases of gramineous plants, in the process of seed germination and germination, It has resistance against bacterial wilt disease or rice blast bacterial disease, and can suppress the onset of these bacterial diseases during seedling raising.

  In addition, after the bacterial disease control agent is coated, the effect is maintained as long as the bacterial disease control agent is coated. For example, seeds coated with the bacterial disease control agent can be distributed as they are.

1. Herbaspirillum sp. (Herbaspirillum sp.) 022S4-11 strain (reception number FERM AP-22001) on rice blast fungus disease (1) Seedling growth conditions Rice seedlings were grown under the following conditions. For seeding, put seedling culture soil (Iseki culture soil) in a plastic case (35mm x 110mm x 110mm) with 5 holes of about 1mm diameter for drainage on the bottom, and uniformly sow 15g treated seed pods to lightly cover the soil did. In addition, after sowing, it managed in the glass greenhouse.

(2) Method for producing contaminated rice shoots Burkholderia glumae
MAFF301441) suspension (approx. 10 ⁸ cfu / ml), seeded with pathogenic bacteria under vacuum decompression for approximately 10 minutes, and then spread the dried seed cake on a lab towel, etc. overnight at room temperature Air dried.

(3) Preparation and treatment method of each treatment solution Herbaspirillum sp. 022S4-11 strain (reception number FERM AP-22001) (hereinafter sometimes simply referred to as “022S4-11 strain”) PPG A culture solution (hereinafter referred to as “culture solution”) that has been shaken and cultured in a medium (25 ° C., 2 days, 100 rpm), and a culture supernatant solution (hereinafter “ A cell suspension obtained by centrifuging the microbial cells obtained by centrifugation and adding the same amount of sterilized water as the supernatant removed by centrifugation (hereinafter referred to as “cell suspension”) Each was prepared as a treatment solution and used in the test.

  FIG. 1 is a diagram showing the treatment steps of this experiment (treatment at germination, 022S4-11 strain, rice blast blight). After soaking seed pods containing contaminated rice blast fungus, the seed pods after soaking are immersed in the culture solution, supernatant, and cell suspension for 16 hours at 32 ° C. It was. Thereafter, seed buds were budding, and the disease-inhibiting effect against rice blast bacterial disease was investigated. As controls, oxolinic acid wettable powder and Trichoderma atrobilede wettable powder were used according to their respective methods of use.

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

(5) Results Table 3 shows the results of examination of the disease-inhibiting effect of 022S4-11 strain on rice blast fungus disease in the treatment at germination.

2. Herbaspirillum sp. 022S4-11 Strain-Inhibiting Effect against Bacterial Rice Blast Disease during Treatment (1) Seedling Conditions Rice seedlings were grown under the same conditions as described in 1 (1) above. It was.

(2) Contamination soot preparation method Contamination soot was produced by the same operation as 1 (2).

(3) Preparation and treatment method of each treatment solution By the same operation as 1 (3) above, a culture solution, a supernatant solution and a cell suspension of the 022S4-11 strain were prepared and used for the test.

  FIG. 2 is a diagram showing the processing steps of this experiment (treatment during soaking, 022S4-11 strain, rice blast bacterial disease). Treatment was performed by immersing seed pods containing contaminated culm of rice blast bacteriomycetes at 25 ° C. for 48 hours in respective treatment solutions of a culture solution, a supernatant solution and a cell suspension. Thereafter, seed buds were soaked, sprouting and budding treatment were performed, and the disease inhibitory effect on rice blast blight was investigated. As controls, oxolinic acid wettable powder and Trichoderma atrobilede wettable powder were used according to their respective methods of use.

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

(5) Results Table 4 shows the results. As an example, FIG. 3 and FIG. 4 show the growth of seedlings about two weeks after sowing. Here, in FIG. 3, A indicates the pathogen inoculation / non-treatment section, B indicates the treatment section at 022S4-11 strain suspension, and C indicates the oxolinic acid treatment section. Moreover, in FIG. 4, A shows the pathogen inoculation / no-treatment group, B shows the 022S4-11 strain culture solution treatment group, and C shows the oxolinic acid treatment group.

3. Herbaspirillum sp. 022S4-11 Strain Prevention Effect against Bacterial Blight of Rice Seedling (1) Seedling Conditions The above 1 except that the contamination rate was 1% inoculation with reduced pressure inoculation Seedlings were grown under the same conditions as shown in (1).

(2) Contaminated cocoon production method Contaminated cocoons were produced by the same operation as 1 (2) above except that the pathogen was Burkholderia plantarii MAFF301723.

(3) Preparation and treatment method of each treatment solution By the same operation as 1 (3) above, a culture solution, a supernatant solution and a cell suspension of the 022S4-11 strain were prepared and used for the test.

  FIG. 5 is a diagram showing the treatment steps of this experiment (treatment at germination, 022S4-11 strain, rice seedling blight). After soaking seed pods containing contaminated culm of rice seedling bacteriomycetes, treatment is performed by immersing the seed pods after soaking in the culture solution, supernatant, and cell suspension for 16 hours at 32 ° C. Went. Thereafter, the seed buds were budding, and the disease-inhibiting effect against rice seedling bacterial disease was investigated. As a control, oxolinic acid wettable powder was used according to the method of use.

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

(5) Results Table 5 shows the results. Moreover, the growth condition of the seedling about 2 weeks after sowing is shown in FIGS. Here, in FIG. 6, A represents the pathogen inoculation / non-treated group, B represents the 022S4-11 strain supernatant germination treatment group, and C represents the oxolinic acid treatment group. In FIG. 7, A represents the pathogen inoculation / non-treatment group, B represents the 022S4-11 strain cell suspension treatment group, and C represents the oxolinic acid treatment group. In FIG. 8, A shows an untreated group, B shows a 022S4-11 strain culture solution treatment group, and C shows an oxolinic acid-treated group.

4). Herbaspirillum sp. (Herbaspirillum sp.) 022S4-11 Strain Prevention Effect against Bacterial Blight of Rice Seedling (1) Seedling Conditions Seedlings were grown under the same conditions as described in 1 (1) above. .

(2) Contaminated cocoon production method Contaminated cocoons were produced by the same operation as 1 (2) above except that the pathogen was Burkholderia plantarii MAFF301723.

(3) Preparation and treatment method of each treatment solution By the same operation as 1 (3) above, a culture solution, a supernatant solution and a cell suspension of the 022S4-11 strain were prepared and used for the test.

  FIG. 9 is a diagram showing the processing steps of this experiment (treatment during soaking, 022S4-11 strain, rice seedling bacterial disease). Treatment was carried out by immersing seed pods containing contaminated culm of rice seedling blight bacteria in each treatment solution of culture solution, supernatant and cell suspension at 25 ° C. for 48 hours. Thereafter, seed buds were soaked, germination and budding treatment were performed, and the disease-inhibiting effect on rice seedling blight was investigated. As controls, oxolinic acid wettable powder and Trichoderma atrobilede wettable powder were used according to their respective methods of use.

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

(5) Results Table 6 shows the results. Moreover, the growth condition of the seedling about two weeks after sowing is shown in FIGS. Here, in FIG. 10, A indicates the pathogen inoculation / non-treatment group, B indicates the 022S4-11 strain supernatant immersion treatment group, and C indicates the oxolinic acid treatment group. In FIG. 11, A represents the pathogen inoculation / non-treatment group, B represents the 022S4-11 strain cell suspension treatment group, and C represents the oxolinic acid treatment group. In FIG. 12, A represents the pathogen-inoculated / non-treated group, B represents the 022S4-11 strain culture-immersion-treated group, and C represents the oxolinic acid-treated group.

5). Herbaspirillum sp. (Herbaspirillum sp.) 022S4-11 Strain Prevention Effect on Rice Brown Stripe Disease (1) Seedling Conditions Seedlings were grown under the same conditions as shown in 1 (1) above.

(2) Contaminated cocoon production method Contaminated cocoons were produced in the same manner as 1 (2) except that the pathogenic bacterium was rice brown streak fungus (Acidovorax avenae MAFF301752).

(3) Preparation and treatment method of each treatment solution By the same operation as 1 (3) above, a culture solution, a supernatant solution and a cell suspension of the 022S4-11 strain were prepared and used for the test.

  FIG. 13 is a diagram showing the treatment process of this experiment (treatment at germination, 022S4-11 strain, rice brown stripe disease). After sowing seed pods containing rice brown stripe fungus contaminated pods, treatment is performed by immersing the seed pods after soaking in a culture solution, a supernatant solution and a cell suspension solution at 32 ° C for 16 hours. It was. Thereafter, the seed buds were budding, and the disease-inhibiting effect on rice brown stripe disease was investigated.

(4) Survey method The degree of disease was investigated for all seedlings in each ward, and an index was given according to the degree. The indices were: dead seedlings: 5, diseased seedlings other than dead seedlings (brown streaks and hip flexion symptoms): 3, healthy seedlings: 0.
Disease severity = {Σ (number of seedlings by degree of disease x index) / (5 x number of seedlings)} x 100
Control value = (1-Disease severity in treated area / Disease severity in untreated area) x 100

(5) Results Table 7 shows the results. 14 to 16 show the growth of seedlings about 2 weeks after the seed pods treated with the treatment solution of the 022S4-11 strain were sown. Here, in FIG. 14, A represents the pathogen inoculation / no treatment group, B represents the 022S4-11 strain culture solution treatment group, and C represents the pathogen inoculation / no treatment group. In FIG. 15, A indicates the pathogen inoculation / non-treatment section, B indicates the treatment area during germination of the cell suspension of the 022S4-11 strain, and C indicates the pathogen inoculation / non-treatment section. In FIG. 12, A represents the pathogen-inoculated / untreated group, B represents the 022S4-11 strain supernatant germination-treated group, and C represents the pathogen-inoculated / untreated group.

6). Herbaspirillum sp. (Herbaspirillum sp.) 022S4-11 Strain Prevention Effect on Rice Brown Stripe Disease (1) Raising conditions Seedling conditions were raised under the same conditions as shown in 1 (1) above.

(2) Contaminated cocoon production method Contaminated cocoons were produced in the same manner as 1 (2) except that the pathogenic bacterium was rice brown streak fungus (Acidovorax avenae MAFF301752).

(3) Preparation and treatment method of each treatment solution By the same operation as 1 (3) above, a culture solution, a supernatant solution and a cell suspension of the 022S4-11 strain were prepared and used for the test.

  FIG. 17 is a diagram showing the treatment process of this experiment (treatment during soaking, 022S4-11 strain, rice brown stripe disease). Treatment was performed by immersing seed pods containing contaminated culm of rice brown streak fungus at 25 ° C. for 48 hours in each of the culture solution, the supernatant solution and the cell suspension. Thereafter, seed buds were soaked, germination and budding treatment were performed, and the disease control effect on rice brown stripe disease was investigated.

(4) Investigation method The diseased seedling rate and disease incidence were calculated by the same calculation method as in 5 (4) above.

(5) Results Table 8 shows the results. 18 to 20 show the growth of seedlings about 2 weeks after the seed pods treated with the treatment solution of the 022S4-11 strain were sown. Here, in FIG. 18, A indicates the pathogen inoculation / no treatment group, B indicates the 022S4-11 strain culture liquid immersion treatment group, and C indicates the pathogen inoculation / no treatment group. In FIG. 19, A represents the pathogen inoculation / no treatment group, B represents the 022S4-11 strain cell suspension treatment group, and C represents the pathogen inoculation / no treatment group. In FIG. 20, A shows the pathogen inoculation / non-treatment section, B shows the 022S4-11 strain supernatant treatment process section, and C shows the pathogen inoculation / non-treatment section.

7). Inhibitory effect of Herbaspirillum rubrisubalbicans MAFF311406 on rice blast bacterial disease during germination treatment (1) Seedling conditions The seedlings were grown under the same conditions as shown in 1 (1) above.

(2) Contamination soot preparation method Contamination soot was produced by the same operation as 1 (2).

(3) Preparation and processing method of each treatment solution Herbaspirillum rubrisubalbicans MAFF311406 strain (hereinafter sometimes simply referred to as “MAFF311406 strain”) is shaken and cultured in PPG medium (25 ° C., 2 A culture solution (hereinafter referred to as “culture solution”) that was 100 rpm per day was prepared as a treatment solution and used for the test.

FIG. 21 is a diagram showing the treatment process of this experiment (treatment at germination, MAFF311406 strain, rice blast blight). After soaking seed pods containing contaminated rice blast fungus, the seed pods were treated by immersing the seed pods in a culture solution at 32 ° C. for 16 hours. Thereafter, seed buds were budding, and the disease-inhibiting effect against rice blast bacterial disease was investigated. As a control, Herbaspirillum sp.
The culture solution of the 022S4-11 strain culture medium was treated.

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

(5) Results Table 9 shows the results. FIG. 22 shows the growth of seedlings about 2 weeks after the seed pods treated with the MAFF311406 strain treatment solution were sown. Here, in FIG. 18, A represents the pathogen inoculation / non-treatment group, B represents the MAFF311406 strain culture solution germination treatment group, and C represents the 022S4-11 strain culture solution germination treatment group.

8). Disease suppression effect of Herbaspirillum rubrisubalbicans MAFF311406 on rice blast blight in treatment during soaking Seedling conditions (1) Seedling conditions The seedlings were grown under the same conditions as shown in 1 (1) above.

(2) Contamination soot preparation method Contamination soot was produced by the same operation as 1 (2).

(3) Preparation and treatment method of each treatment solution By the same operation as 7 (3) above, a culture solution, a supernatant and a cell suspension of MAFF311406 strain were prepared and used for the test.

  FIG. 23 is a diagram showing the processing steps of this experiment (treatment during soaking, MAFF311406 strain, rice blast blight). Treatment was carried out by immersing seed pods containing contaminated culm of rice blast bacteriomycetes for 48 hours at 25 ° C. in each of the culture solution, supernatant solution and cell suspension. Thereafter, seed buds were soaked, sprouting and budding treatment were performed, and the disease inhibitory effect on rice blast blight was investigated. As a control, a culture solution, a supernatant solution and a cell suspension of Herbaspirillum sp. 022S4-11 were treated at the time of seeding.

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

(5) Results The results are shown in FIGS. 24 to 26 show the growth of seedlings about 2 weeks after the sowing of the seed pods treated with the treatment solution of MAFF311406 strain. Here, in FIG. 24, A represents the pathogen inoculation / non-treatment group, B represents the MAFF311406 strain culture-immersion treatment group, and C represents the 022S4-11 strain culture-immersion treatment group. In FIG. 25, A represents the pathogen-inoculated / untreated group, B represents the MAFF311406 strain supernatant immersion treatment group, and C represents the 022S4-11 strain supernatant immersion treatment group. In FIG. 26, A represents the pathogen inoculation / non-treatment group, B represents the MAFF311406 strain cell suspension treatment period, and C represents the 022S4-11 strain suspension seed treatment group.

9. Herbaspirillum rubrisubalbicans MAFF311406 strain suppression effect on rice seedling bacterial disease during germination treatment (1) Seedling conditions 1 Seedlings were grown under the same conditions as shown in (1).

(2) Contaminated cocoon production method Contaminated cocoons were produced by the same operation as 7 (2) except that the pathogen was Burkholderia plantarii MAFF301723.

(3) Preparation and treatment method of each treatment solution A culture solution of MAFF311406 strain was prepared by the same operation as 7 (3) above and used for the test.

FIG. 27 is a diagram showing the processing steps of this experiment (treatment during germination, MAFF311406 strain, rice seedling blight). After immersing seed pods containing contaminated culm of rice seedling blight, the treatment was performed by immersing them in a treatment solution at 32 ° C. for 16 hours. Thereafter, budding treatment was carried out, and the disease-suppressing effect against rice seedling bacterial disease was investigated. As a control, Herbaspirillum sp.
The culture solution of the 022S4-11 strain culture medium was treated.

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

(5) Results Table 10 shows the results. FIG. 28 shows the growth of seedlings about 2 weeks after the sowing of the seed pods treated with the treatment solution of MAFF311406 strain. Here, in FIG. 28, A represents the pathogen inoculation / non-treatment group, B represents the MAFF311406 strain culture solution germination treatment group, and C represents the 022S4-11 strain culture solution germination treatment group.

10. Herbaspirillum rubrisubalbicans MAFF311406 strain suppression effect on rice brown streak in treatment during soaking Seedling conditions (1) Seedling conditions The seedlings were grown under the same conditions as shown in 1 (1) above.

(2) Contaminating cocoon production method Contaminating cocoon was produced by the same operation as said 7 (2) except that the pathogenic bacterium is rice brown streak fungus (Acidovorax avenae MAFF301752).

(3) Preparation and treatment method of each treatment solution A culture solution of MAFF311406 strain was prepared by the same operation as 7 (3) above and used for the test.

  FIG. 29 is a diagram showing the processing steps of this experiment (treatment during soaking, MAFF311406 strain, rice brown stripe disease). Treatment was carried out by immersing seed pods containing contaminated culm of rice brown stripe fungus in a treatment solution at 25 ° C. for 48 hours. Thereafter, seed buds were soaked, sprouting and budding treatment were performed, and the disease control effect on rice brown stripe disease was investigated.

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

(5) Results Table 11 shows the results. FIG. 30 shows the growth of seedlings about 2 weeks after the sowing of the seed pods treated with the treatment solution of MAFF311406 strain. Here, in FIG. 30, A represents the pathogen inoculation / no treatment group, B represents the 022S4-11 strain culture solution immersion treatment group, and C represents the MAFF311406 strain culture immersion treatment group.

11. Inhibitory effect of Herbaspirillum spp. On rice bacteriomycetes in treatment during soaking (1) Seedling conditions Rice seedlings were grown under the following conditions. For seeding, place seedling culture soil (Iseki culture soil) in a plastic case (15mm x 44mm x 44mm) with 5 holes of about 1mm in diameter for drainage on the bottom, and uniformly seed 3g treated seed cake to lightly cover the soil did. In addition, after sowing, it managed in the glass greenhouse. Others were grown under the conditions shown in Table 2.

(2) Contamination soot preparation method Contamination soot was produced by the same operation as 2 (2).

(3) Preparation and treatment method of each treatment solution The above-mentioned bacteria belonging to the genus Herbaspirillum are Herbaspirillum autotrophicum, Herbaspirillum chlorophenolicum, Herbaspirillum chlorophenolicum, Except for Frissingense (Herbaspirillum frisingense), Herbaspirillum huttiense, Herbaspirillum putei, Herbaspirillum seropedicae, the same operation as 2 (3) above A culture solution, a supernatant and a cell suspension of Herbaspirillum bacteria were prepared and used for the test. As a control, a section (pathogen inoculation / non-treatment section) was prepared by immersing contaminated sputum in distilled water instead of the treatment liquid and other conditions.

(4) Investigation method The diseased seedling rate and disease incidence were calculated by the same calculation method as in 2 (4) above.

(5) Results The results are shown in Tables 12-17. As an example, FIG. 31 shows the growth of seedlings about 2 weeks after seeding after treatment with the culture solution, and FIG. 32 shows seedlings about 2 weeks after seeding with the supernatant treatment. FIG. 33 shows the growth situation of seedlings about 2 weeks after the seeding after the cell suspension treatment. In FIGS. 31 to 33, A represents the pathogen inoculation / non-treatment section, B represents the treatment section at the time of the soaking of the Herbus pyriram petit strain, and C represents the treatment section at the time of the soaking of the 022S4-11 strain (reference).

12 Disease suppression effect on rice seedling blight disease of Herbaspirillum genus fungus in treatment during soaking (1) Seedling conditions Rice seedlings were grown under the same conditions as shown in 11 (1) above.

(2) Contamination soot preparation method Contamination soot was produced by the same operation as said 4 (2).

(3) Preparation and treatment method of each treatment solution The above-mentioned bacteria belonging to the genus Herbaspirillum are Herbaspirillum autotrophicum, Herbaspirillum chlorophenolicum, Herbaspirillum chlorophenolicum, Except for Hrybaspirillum frisingense, Herbaspirillum huttiense, Herbaspirillum putei, Herbaspirillum seropedicae, the same operation as 4 (3) above A culture solution, a supernatant and a cell suspension of Herbaspirillum bacteria were prepared and used for the test. As a control, a section (untreated section) was prepared by immersing contaminated soot in distilled water instead of the treatment solution and investigating other conditions.

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

(5) Results The results are shown in Tables 18-23.

13. Disease control effect on rice brown streak of Herbaspirillum spp. In the treatment at the time of soaking (1) Seedling conditions Seedlings were grown under the same conditions as shown in 6 (1) above.

(2) Contamination soot preparation method Contamination soot was produced by the same operation as 6 (2).

(3) Preparation and treatment methods of each treatment solution The genus bacteria of Herbaspirillum are Herbaspirillum autotrophicum, Herbaspirillum chlorophenolicum, Herbaspirillum chlorophenolicum, Except for Herbaspirillum frisingense, Herbaspirillum huttiense, Herbaspirillum putei, and Herbaspirillum seropedicae, the same operation as 6 (3) above A culture solution, a supernatant and a cell suspension of Herbaspirillum bacteria were prepared and used for the test.

(4) Investigation method The diseased seedling rate and disease incidence were calculated by the same calculation method as in 6 (4).

(5) Results The results are shown in Tables 24-29.

14 Growth-promoting effect of Herbaspirillum sp. 022S4-11 on rice seedlings (1) Materials and methods Seeds of Kojihikari inoculated with 022S4-11 isolated from rice leaf sheath as follows The effect of rice seedlings on growth was investigated. First, the seed pod was immersed in tap water at 20 ° C. for 3 days to cause the seed pod to absorb water. The tap water was changed every day.

Next, after culturing 022S4-11 strain in PPGA (potato, peptone, glucose, agar) medium for 2 days, the cells obtained with a centrifuge were suspended in sterile water to a concentration of about 10 ⁸ cfu / ml. A cell suspension adjusted to 1 was prepared.

  After seeding the above seed pods at 25 ° C for 3 days, the seed pods were put into the cell suspension, and the seed pods were immersed for 24 hours while shaking (50 rpm / min) at 30 ° C, so that 022S4-11 strain was added to the seed pods. Adhered (treatment at germination). Thereafter, the seed pods were sown and cultivated in a seedling culture soil. In addition, as a control, the treatment with the cell suspension was not performed at the time of germination, the germination treatment was performed at 30 ° C. for 24 hours by a conventional method, and the seed buds subjected to each treatment of budding were sown on the seedling culture soil and cultivated.

  For seeding, place seedling culture soil (Sumitomo Chemical Bonsol No. 1) in a plastic case (3.5mm x 10mm x 10mm) with 5 holes with a diameter of about 1mm for drainage at the bottom, and add 10g of treated seed cake. Seeded uniformly and covered lightly. Cultivation was carried out in a glass greenhouse, and the fresh weight and plant height were investigated 14 days after sowing.

(2) Results Table 30 shows the results. FIG. 34 shows the growth of seedlings about two weeks after the seed pods treated with the cell suspension of the 022S4-11 strain were sown. Here, in FIG. 34, A represents the treatment group during germination of the 022S4-11 strain cell suspension, and B represents the non-treatment group.

  In the group treated with the cell suspension of the 022S4-11 strain at the time of germination (in the table, indicated as “treatment group at the time of germination of the 022S4-11 strain”), the growth of rice seedlings was promoted compared to the untreated group. Turned out to be. Further, when the presence or absence of nitrogen fixation ability was examined by the acetylene reduction method, nitrogen fixation ability was confirmed in 022S4-11 strain. Therefore, it was speculated that the factor that promoted the growth of rice seedlings in the group treated with the cell suspension of the 022S4-11 strain at the time of germination was the nitrogen fixing ability of the 022S4-11 strain.

Claims (19)

  A bacterial disease control agent that contains a culture solution of a genus Herbaspirillum or a supernatant thereof, and is effective in controlling bacterial diseases of gramineous plants.   The bacterium belonging to the genus Herbaspirillum is at least one selected from the group consisting of Herbaspirillum sp. And Herbaspirillum rubrisubalbicans. Bacterial disease control agent as described in.   The bacterium belonging to the genus Herbaspirillum is Herbaspirillum autotrophicum, Herbaspirillum chlorophenolicum, Herbaspirillum frisingense, Herbaspirillum frisingense, The bacterial disease control agent according to claim 1, which is at least one selected from the group consisting of (Herbaspirillum huttiense), Herbaspirillum putei, and Herbaspirillum seropedicae.   The bacterial disease control agent according to claim 1 or 2, wherein the Herbaspirillum genus bacterium is Herbaspirillum sp. 022S4-11 strain (accession number FERM AP-22001).   The bacterial disease control agent according to any one of claims 1 to 4, wherein the bacterial disease is a rice seedling bacterial disease or a rice foliage bacterial disease.   A bacterial disease control agent comprising a genus Herbaspirillum or a crushed product thereof, and effective in controlling bacterial diseases of gramineous plants.   The bacterial disease control agent according to claim 6, wherein the genus Herbaspirillum is at least one selected from the group consisting of Herbaspirillum sp.   The bacterium belonging to the genus Herbaspirillum is Herbaspirillum autotrophicum, Herbaspirillum chlorophenolicum, Herbaspirillum frisingense, Herbaspirillum frisingense, The bacterial disease control agent according to claim 6, which is at least one selected from the group consisting of (Herbaspirillum huttiense), Herbaspirillum putei, and Herbaspirillum seropedicae.   The bacterial disease control agent according to claim 6 or 7, wherein the genus Herbaspirillum bacterium is Herbaspirillum sp. Strain 022S4-11 (accession number FERM AP-22001).   The bacterium according to any one of claims 6 to 9, wherein the bacterial disease is at least one selected from the group consisting of rice seedling blight disease, rice blast bacterial disease, and rice brown streak disease. Disease control agent.   The control method of the bacterial disease of the Gramineae plant which has the control process process which makes the seed of Gramineae plant adhere to the bacterial disease control agent of any one of Claims 1-5.   The method for controlling bacterial diseases of gramineous plants according to claim 11, wherein the control treatment step is a step of immersing the seed in the bacterial disease control agent.   The control method of the bacterial disease of the Gramineae plant which has the control process process which makes the seed of Gramineae plant adhere to the bacterial disease control agent of any one of Claims 6-10.   The method for controlling bacterial diseases of gramineous plants according to claim 13, wherein the control treatment step is a step of immersing the seed in the bacterial disease control agent.   Herbaspirillum sp. 022S4-11 strain (reception number FERM AP-22001).   A bacterial disease control agent effective for controlling bacterial diseases of gramineous plants, comprising a Herbaspirillum genus bacterium or a disrupted product thereof or a culture solution of Herbaspirillum genus bacterium or a supernatant thereof. Seeds coated on grass seeds.   The bacterium belonging to the genus Herbaspirillum is at least one selected from the group consisting of Herbaspirillum sp. And Herbaspirillum rubrisubalbicans. Seeds described in.   The bacterium belonging to the genus Herbaspirillum is Herbaspirillum autotrophicum, Herbaspirillum chlorophenolicum, Herbaspirillum frisingense, Herbaspirillum frisingense, The seed according to claim 16, wherein the seed is at least one selected from the group consisting of (Herbaspirillum huttiense), Herbaspirillum putei, and Herbaspirillum seropedicae.   The seed according to claim 16 or 17, wherein the bacterium belonging to the genus Herbaspirillum is Herbaspirillum sp. 022S4-11 strain (accession number FERM AP-22001).