Agent for controlling potato blackleg disease

Abstract

To provide means for controlling blackleg disease through the use of a plant endophyte.SOLUTION: A potato blackleg disease control agent includes a microorganism that belongs to Agrobacterium fabrum and exhibits the ability to control potato blackleg disease.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a potato black leg disease control agent, a potato black leg disease control method, a potato cultivation method, and a bacterial strain capable of controlling potato black leg disease. [Background technology]

[0002] Potato black leg disease (hereafter simply referred to as "black leg disease") is a bacterial disease that spreads to seed potatoes and is one of the most serious diseases in seed potato production. To prevent contamination with infectious pathogenic bacteria and viruses, seed potatoes are produced under strict cultivation management, starting at the Seed Management Center. However, in 2014, black leg disease occurred in the Seed Management Center's original seed production field, shaking the foundation of stable seed potato production. To prevent black leg disease, cultural measures such as maintaining appropriate crop rotation, improving field drainage, using disease-free seed potatoes, early detection and removal of diseased plants, and drying the surface of seed potatoes after harvest are used, as well as chemical measures such as disinfecting seed potatoes with fungicides and disinfecting cutting knives. Seed potato disinfection is effective in killing the black leg disease fungus present on the surface of seed potatoes, but in recent years it has become clear that all black leg disease fungus species are also carried inside seed potatoes. Seed potato disinfection is ineffective against black leg disease fungus carried inside, making control difficult. Furthermore, the Green Food System Strategy established by the government in 2021 aims to reduce the use of chemical pesticides (risk equivalent) by 50%, and there are hopes for the development of new disease control technologies that utilize control methods such as plant endophytes.

[0003] Since the pathogen of potato black leg disease (black leg fungus) is known to be present mainly on the surface of seed potatoes, such as in soil attached to the surface of the seed potatoes, in wounds on the epidermis, and in the lenticels, disinfection of seed potatoes by immersion in a fungicide before planting is practiced to prevent the disease from spreading to seed potatoes. Specifically, for example, seed potatoes are immersed in a 60- to 100-fold dilution of streptomycin solution for 5 to 10 seconds before planting (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] FY2022 Hokkaido Crop Disease, Pest and Weed Control Guide, Hokkaido Prefectural Agriculture Department Summary of the Invention [Problem to be solved by the invention]

[0005] The fungicide immersion treatment described above is effective in killing the black leg disease fungus present on the surface of seed potatoes and is a simple process, but it is not effective against the fungus that is carried internally. Furthermore, fungicides that contain antibiotics may cause drug-resistant strains of the pathogen (Takahashi, 1977).

[0006] As such, there are many problems with controlling black leg disease using fungicides. Using plant endophytes instead of fungicides is expected to solve these problems. Research on disease control using plant endophytes has been conducted in Japan. Examples include the use of Pseudomonas fluorescens to control bacterial wilt in tomato (Aino et al., 1997) and Heteroconium chaetospira to control clubroot disease in cruciferous vegetables (Narisawa et al., 2000). While there have been reports of research on potato endophytes (Reiter et al., 2002), no examples have been reported of identifying endophytes capable of suppressing black leg disease and linking them to disease control.

[0007] The present invention has been made under the above-mentioned circumstances, and aims to provide a means for controlling black leg disease using a plant endophyte. [Means for solving the problem]

[0008] As a result of extensive research to solve the above problems, the present inventors discovered that a microorganism belonging to Agrobacterium fabrum has the ability to control black leg disease, and based on this finding, they have completed the present invention.

[0009] That is, the present invention provides the following [1] to [6]. [1] A potato black leg disease control agent characterized by containing a microorganism belonging to Agrobacterium fabrum and having the ability to control potato black leg disease.

[0010] [2] The potato black leg disease control agent according to [1], characterized in that the microorganism belonging to Agrobacterium fabrum and having the ability to control potato black leg disease is Agrobacterium fabrum 6F10 strain (received number: NITE AP-04069), Agrobacterium fabrum 13A3 strain (received number: NITE AP-04070), or Agrobacterium fabrum 13C4 strain (received number: NITE AP-04071).

[0011] [3] A method for controlling potato black leg disease, comprising the step of immersing potato tubers in a liquid containing the potato black leg disease control agent according to [1] or [2].

[0012] [4] The method for controlling potato black leg disease according to [3], wherein the tubers are tubers for seed potatoes.

[0013] [5] A method for cultivating potatoes, comprising the following steps (1) to (3): (1) A step of immersing potato tubers in a liquid containing the potato black leg disease control agent according to [1] or [2]; (2) after step (1), planting potato tubers in a field; (3) After step (2), growing potatoes.

[0014] [6] Agrobacterium fabrum 6F10 strain (received number: NITE AP-04069), Agrobacterium fabrum 13A3 strain (received number: NITE AP-04070), or Agrobacterium fabrum 13C4 strain (received number: NITE AP-04071). [Effects of the Invention]

[0015] The present invention provides a novel control agent for potato black leg disease. The control agent of the present invention can control potato black leg disease by simply immersing seed potatoes in the same manner as previously used fungicides. Furthermore, because the control agent of the present invention utilizes an endophyte, it also contributes to reducing the amount of chemical pesticides used. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention will be described in detail below. The potato black leg disease control agent of the present invention is characterized by containing a microorganism belonging to Agrobacterium fabrum and having the ability to control potato black leg disease.

[0017] Examples of the microorganisms used include Agrobacterium fabrum 6F10, Agrobacterium fabrum 13A3, and Agrobacterium fabrum 13C4. These strains have been deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation. Detailed information on the deposit is as follows:

[0018] (1) Agrobacterium fabrum 6F10 strain (1-1) Name and address of the depository institution Name: National Institute of Technology and Evaluation, Patent Microorganism Deposit Center Address: 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan Postal code: 292-0818 (1-2)Received date: January 25, 2024 (1-3) Receipt number: NITE AP-04069 (1-4) Identification: Agrobacterium fabrum 6F10

[0019] (2) Agrobacterium fabrum 13A3 strain (1-1) Name and address of the depository institution Name: National Institute of Technology and Evaluation, Patent Microorganism Deposit Center Address: 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan Postal code: 292-0818 (1-2)Received date: January 25, 2024 (1-3) Receipt number: NITE AP-04070 (1-4) Identification: Agrobacterium fabrum 13A3

[0020] (3) Agrobacterium fabrum 13C4 strain (1-1) Name and address of the depository institution Name: National Institute of Technology and Evaluation, Patent Microorganism Deposit Center Address: 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan Postal code: 292-0818 (1-2)Received date: January 25, 2024 (1-3) Receipt number: NITE AP-04071 (1-4) Identification: Agrobacterium fabrum 13C4

[0021] Microorganisms other than the above strains can be used as long as they belong to the genus Agrobacterium fabram and have the ability to control potato black leg disease. As described in the Examples below, such microorganisms can be obtained by isolating endophytic bacteria (endophytes) from potatoes, selecting from the isolated strains those that have the effect of suppressing tuber disc rot or black leg disease, and then selecting from these strains those that belong to Agrobacterium fabram. Microorganisms that can be used in the present invention can also be obtained by selecting from known strains that belong to Agrobacterium fabram those that have the effect of suppressing tuber disc rot or black leg disease.

[0022] The microorganisms used in the present invention can be cultured by any method commonly used for Agrobacterium fabram. For example, LB liquid medium can be used. The culture temperature can be around 25°C (e.g., 20-30°C).

[0023] The content of the microorganism contained in the control agent of the present invention is not particularly limited as long as it is within a range that allows for the control of potato black leg disease. 6 ~10 12 cfu / ml, preferably 10 8 ~10 10 It can be expressed as cfu / ml.

[0024] The control agent of the present invention may be a simple suspension of the microorganism described above in water, or it may be formulated in combination with other optional ingredients into an aqueous suspension, stabilized liquid suspension, emulsion, capsule, soluble powder, wettable powder, aqueous flowable, dry flowable, water dispersible granule, water dispersible granule, etc. Optional ingredients used in combination include, for example, diluents and carriers. Diluents include monosaccharides, polysaccharides, molasses, gum, lignosulfonates, glycerin, sorbitol, propylene glycol, water, vegetable oil, mineral oil, etc. Carriers include alginate beads, starch, granules, silica, clay, clay minerals (e.g., attapulgite, kaolinite, montmorillonite, pyrophyllite, illite), gelatin, cellulose, cellulose derivatives, calcium chloride, talc powder, etc.

[0025] The disease targeted for control is potato black leg disease, a bacterial disease transmitted to seed potatoes. After seed potatoes germinate, the seed potatoes first rot, and then the rot spreads to the sprout stems, causing black leg symptoms. The causative agents of potato black leg disease are known to be Pectobacterium atrosepticum, Pectobacterium wasabiae, Pectobacterium carotovorum subsp. brasiliense, Dickeya dianthicola, and Dickeya chrysanthemi.

[0026] The target plants of the control agent are plants that develop potato black leg disease, usually potatoes (Solanum tuberosum).

[0027] The method for controlling potato black leg disease of the present invention uses the potato black leg disease control agent of the present invention and is characterized by including a step of immersing potato tubers in a liquid containing the control agent. Potato tubers are usually tubers for seed potatoes. The concentration of microorganisms in the solution for soaking the tubers is not particularly limited as long as it is a concentration that can control potato black leg disease. For example, 6 ~10 12 cfu / ml, preferably 10 8 ~10 10 cfu / ml. The immersion time can be very short, for example, 1 to 30 seconds, preferably 5 to 10 seconds. After the immersion treatment, the tubers may be planted in a field immediately, but usually the surface of the seed potatoes is dried before planting.

[0028] By using the potato black leg disease control agent of the present invention, a potato cultivation method that prevents the occurrence of potato black leg disease can be provided, for example, a method comprising the following steps (1) to (3). (1) A step of immersing potato tubers in a liquid containing the potato black leg disease control agent of the present invention. (2) After step (1), planting potato tubers in the field. (3) After step (2), growing potatoes. [Example]

[0029] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0030] Materials and Methods 1. Test strains Potato field soil collected from four locations in Hokkaido (three in Yakumo Town, Futami District, and one in Imakane Town, Setana District) between 2003 and 2004 was suspended in hydroponic culture solution (Table 1). Roots of potato meristem seedlings (variety: Danshaku potato), with their root tips removed, were immersed in the suspension and grown at 22°C for approximately one month. The roots were washed with tap water and then with sterile water, then ground. Eight hundred seventy-two bacterial strains were isolated from the root washings and the root grounds by the dilution plating method using YG medium. The isolated bacteria were suspended in 10% glycerol and stored frozen at -80°C. For use, they were cultured in LB liquid medium at 25°C with shaking at 120 rpm for 48–96 hours. After growth, the bacteria were harvested and washed. [Table 1]

[0031] 2. Screening [1] Tuber disc rot suppression test (2022) Disks were punched out of potato tuber slices (5 mm thick) using a cork borer (inner diameter 14 mm) and placed individually in a 24-well multiwell plate. A water suspension of the test strain (ca. 10 8 cfu / ml) was added to the plants and pre-inoculated. Then, a suspension of Pcb. kbs-1 strain (ca. 10 6 cfu / disc) was inoculated onto tuber discs in an amount of 10 μl, the plates were covered, and cultured in a moist chamber at 25°C. After 48 hours of culture, the tuber discs were inspected for decay and the decay inhibitory effect was evaluated. Three replicates (3 discs / strain) were performed for each strain, with a group that was not inoculated with either the test strain or the blackleg disease fungus (uninoculated group), and a group that was inoculated with the blackleg disease fungus alone (untreated group).

[0032] 3. Screening [2] Black leg disease suppression test (Pot, 2022) Blackleg fungus suspension (Pcb kbs-1 strain ca. 10 6 Seed potatoes (Toyoshiro) were immersed in a suspension of the test strain (ca. 10 cfu / ml) for 15 minutes under reduced pressure (-0.06 MPa), and then air-dried at room temperature to prepare tubers inoculated with the fungus (hereafter referred to as inoculated tubers). 9cfu / ml) were instantly immersed in the tubers and then air-dried at room temperature. The tubers inoculated with the test strain were planted in polypots filled with Andosol and cultivated in a glass greenhouse (room temperature 20-30°C), with observations made at regular intervals for germination and blackleg disease development. Five replicates were performed for each strain, with an uninoculated area (neither the test strain nor the blackleg disease fungus was inoculated), an untreated area (inoculated with the blackleg disease fungus only), and an insecticide-treated area (tubers from the untreated area were instantly immersed in a 500x dilution of Agrepto solution).

[0033] 4. Screening [3] Black leg disease suppression test (Field, 2023) Blackleg fungus suspension (Pcb kbs-1 strain ca. 10 6 Seed potatoes (Toyoshiro) were immersed in a suspension of the test strain (ca. 10 cfu / ml) for 15 minutes under reduced pressure (-0.06 MPa), and then air-dried at room temperature to prepare tubers inoculated with the fungus (hereafter referred to as inoculated tubers). 9 cfu / ml) were instantly immersed in the tubers and then air-dried at room temperature. The tubers inoculated with the test strains were planted in a field at the Hokkaido Agricultural Research Center in Hitsujigaoka, Toyohira Ward, Sapporo, and cultivated according to conventional methods. The germination and incidence of black leg disease were monitored at regular intervals. Each strain was tested in three replicates, with 12 strains per plot. As with screening [2], there were uninoculated, untreated, and fungicide-treated plots.

[0034] 5. Identification of effective bacterial strains 1) Nucleotide sequence analysis of the 16S ribosomal DNA (rDNA) region (1) DNA extraction The test strains were cultured in LB liquid medium, and bacterial DNA was extracted and purified from the cultured cells using NucleoSpin Tissue (Machrei-Nagel). The resulting DNA solution was adjusted to 20 ng / μl with 5 mM Tris-HCl (pH 8.5).

[0035] (2) PCR amplification of the 16S rDNA region The PCR reaction mixture (25 μl) was prepared according to the method described by Suharjo et al. (2007). Specifically, 12.50 μl of DNA polymerase master mix (AmpliTaq Gold 360 Master Mix, Thermo Fisher Scientific) was used, 0.63 μl each of forward primer (fD1, 20 μM; 5′-CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG-3′ (SEQ ID NO: 1)) and reverse primer (rP2, 20 μM; 5′-CCCGGGATCCAAGCTTACGGCTACCTTGTTACGACTT-3′ (SEQ ID NO: 2)), 4 μl of test strain DNA solution, and 7.25 μl of ultrapure water. PCR reactions were performed using an Applied Biosystems SimpliAmp Thermal Cycler (Thermo Fisher Scientific) under the following reaction conditions: [1] a hot start at 94°C for 5 minutes; [2] 35 amplification cycles of 94°C for 1 minute, 58°C for 1 minute, and 72°C for 1 minute; and [3] a final extension reaction at 72°C for 7 minutes. The PCR reaction mixture was electrophoresed (100 V, approximately 30 minutes) on a 2% agarose gel in 0.5x Tris-borate-EDTA buffer (TBE). The gel was then stained for 1 hour in 0.5x TBE containing Midori Green Advance (1 μl / 100 ml, Nippon Genetics). The amplification product band was excised on an LED transilluminator, and the PCR amplification product was purified using FastGene Gel / PCR Purification (Nippon Genetics). The DNA concentration was measured using a spectrophotometer and adjusted to 20 ng / μl with 5 mM Tris-HCl (pH 8.5).

[0036] (3) Sequence analysis of the 16S rDNA region Sequencing reactions were performed according to the manufacturer's protocol. A 20-μl PCR reaction mixture was prepared containing 1 μl of BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Mix (Thermo Fisher Scientific), 3.5 μl of BigDye Terminator v1.1 & v3.1 5X Sequencing Buffer (Thermo Fisher Scientific), 0.16 μl of forward primer (fD1, 20 μM) or reverse primer (rP2, 20 μM), 1 μl of the PCR amplification product solution (2), and 14.34 μl of ultrapure water. The PCR reaction mixture was then subjected to 25 cycles of denaturation at 96°C for 1 minute, amplification at 96°C for 10 seconds, 50°C for 5 seconds, and 72°C for 4 minutes using an Applied Biosystems SimpliAmp Thermal Cycler (Thermo Fisher Scientific). The sequencing reaction solution was purified using the FastGene Dye Terminator Removal Kit (Nippon Genetics), and 20 μl (total volume) of the purified product was subjected to sequencing using a SeqStudio Genetic Analyzer (Thermo Fisher Scientific).

[0037] (4) Homology search using BLAST The obtained nucleotide sequence data of the 16S rDNA region of the test strains was subjected to a homology search with sequence information in the database using the BLAST service provided by the National Center for Biotechnology Information (NCBI). The sequence information was registered with the DNA Data Bank of Japan (DDBJ) under accession numbers LC799031 to LC799033.

[0038] 2) Bacteriological analysis using API20 NE Cultured cells of the test strain were suspended in 1 ml of 0.85% saline and adjusted to a McFarlane turbidity of 0.5. The prepared bacterial suspension was used to identify non-enterobacterial Gram-negative bacilli using the API20NE (Sysmex bioMérieux) identification kit, according to the manufacturer's instructions. After culturing under the specified conditions, positive / negative results were determined based on the reaction to each substrate, and the resulting 7-digit profile of the strain was compared with a database to identify the bacterial species.

[0039] result 1. Screening [1] Tuber disc rot suppression test (2022) Selection was based on the criterion that none of the three discs were decayed, and 28 strains that met this criterion were selected from the 872 strains.

[0040] 2. Screening [2] Black leg disease suppression test (Pot, 2022) Selection was based on the following criteria: 1) germination in three or more pots, 2) no black leg disease was observed, and 3) the next generation tubers were infected with the virus in 0 to 2 pots. From the 27 strains, 13 strains that met these criteria were selected (Table 2). [Table 2]

[0041] 3. Screening [3] Black leg disease suppression test (Field, 2023) Five strains were selected from the 13 strains selected in the screening [2], and the control value was calculated from the diseased plant rate. Three strains that met the A or B criteria in the overall evaluation (Japan Plant Protection Association's new pesticide practical application test evaluation criteria) were selected (Table 3). [Table 3]

[0042] 4. Homology search results for the 16S ribosomal DNA region base sequence The 16S rDNA sequences of 6F10 and 13C4, two of the three strains selected in screening [3], and 13A3, one of the 13 strains selected in screening [2], were examined. The 16S rDNA sequences of these strains all showed extremely high homology (99.87-100%) with the registered sequence of Agrobacterium fabrum 12D13 strain in the database (accession number CP033035.1) (Table 4). [Table 4]

[0043] 5. Identification of test strains based on bacteriological analysis results using the API20NE kit Bacteriological analysis of the above three strains 6F10, 13A3, and 13C4 using the API20NE kit showed that they corresponded to Rhizobium radiobacter with a very high probability (97.8 to 99.8%) (Table 5). [Table 5]

[0044] 6. Identification of test strains Rhizobium radiobacter, shown in 5, was originally identified as Agrobacterium tumefaciens, and A. fabrum was established by upgrading A. tumefaciens genomovar G8 to a species (Lassalle et al., 2011). Based on this and the results of a homology search using the 16S rDNA base sequence, the test strains 6F10, 13A3, and 13C4 were identified as A. fabrum.

[0045] References Aino, M., Maekawa, Y., Mayama, S. and Kato, H. (1997) Biocontrol of bacterial wilt of tomato by producing seedlings colonized with endophytic antagonistic pseudomonads. In: Proceedings of the 4th International Workshop on Plant Growth-Promoting Rhizobacteria, Japan-OECD Joint Workshop, Sapporo, pp. 120-123. Lassalle, F., Campillo, T., Vial, L., Baude, J., Costechareyre, D., Chapulliot, D., Shams, M., Abrouk, D., Lavire, C., Oger-Desfeux, C., Hommais, F., Gueguen, L., Daubin, V., Muller, D. and Nesme, X. (2011) Genomic species are ecological species as revealed by comparative genomics in Agrobacterium tumefaciens. Genome Biol. Evol. 3:762-81. doi: 10.1093 / gbe / evr070. Merz, U. (1989) Infectivity, inoculum density and germination of Spongospora subterranea resting spores: a solution-culture test system. Bull. OEPP 19:585-592. Narisawa, K., Ohki, T. and Hashiba, T. (2000) Suppression of clubroot and Verticillium yellows in Chinese cabbage in the field by the root endophytic fungus, Heteroconium chaetospira, Plant Pathology 49: 141-146. Reiter, B., Pfeifer, U., Schwab, H. and Sessitsch, A. (2002) Response of Endophytic Bacterial Communities in Potato Plants to Infection with Erwinia carotovora subsp. atroseptica, Appl. Env. Microbiol. 69: 2261-2268. Suharjo, R., Sawada, H., Takikawa, Y. (2014) Phylogenetic study of Japanese Dickeya spp. and development of new rapid identification methods using PCR-RFLP. J. Gen. Plant Pathol. 80:237-254. Takahashi, K. (1975) Emergence and countermeasures for drug-resistant plant pathogenic bacteria, Plant Protection 29: 199-205. [Industrial Applicability]

[0046] The present invention can be used in industrial fields such as agriculture.

Claims

1. A potato black leg disease control agent characterized by containing a microorganism belonging to Agrobacterium fabrum and having the ability to control potato black leg disease.

2. 2. The potato black leg disease control agent according to claim 1, wherein the microorganism belonging to Agrobacterium fabrum and having the ability to control potato black leg disease is Agrobacterium fabrum 6F10 strain (received number: NITE AP-04069), Agrobacterium fabrum 13A3 strain (received number: NITE AP-04070), or Agrobacterium fabrum 13C4 strain (received number: NITE AP-04071).

3. A method for controlling potato black leg disease, comprising the step of immersing potato tubers in a liquid containing the potato black leg disease control agent according to claim 1 or 2.

4. 4. The method for controlling potato black leg disease according to claim 3, wherein the tubers are seed tubers.

5. A method for cultivating potatoes, comprising the following steps (1) to (3): (1) a step of immersing potato tubers in a liquid containing the potato black leg disease control agent according to claim 1 or 2; (2) after step (1), planting potato tubers in a field; (3) After step (2), growing potatoes.

6. Agrobacterium fabrum 6F10 strain (received number: NITE AP-04069), Agrobacterium fabrum 13A3 strain (received number: NITE AP-04070), or Agrobacterium fabrum 13C4 strain (received number: NITE AP-04071).

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