Agent for controlling soybean black root rot fungus

Abstract

To provide a new agent for controlling soybean black root rot fungus and to provide a Calonectria ilicicola strain for use in the soybean black root rot fungus control agent.SOLUTION: An agent for controlling soybean black root rot fungus according to one embodiment contains an attenuated Calonectria ilicicola as an active ingredient. A Calonectria ilicicola according to one embodiment is a Calonectria ilicicola strain UH2-1A having the accession number NITE-AP-04095 (deposition number: NITE P-04095).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an agent for controlling soybean black root rot fungus. The present invention also relates to a novel Calonectria irishicola strain, Calonectria irishicola UH2-1A strain (Accession Number: NITE-AP-04095). [Background technology]

[0002] Soybean black root rot is a soil-borne disease caused by Calonectria ilicicola. Because soybean black root rot primarily affects the roots of soybean plants, early detection of infection and disease onset is difficult. Currently, there are no soybean varieties resistant to the disease, and although three registered pesticides exist, the effectiveness of soil spraying is limited. The main countermeasures for soybean black root rot include agricultural control, such as draining fields, converting fields into paddy fields, and temporarily converting converted paddy fields back to paddy fields, but these methods are only effective. Therefore, the development of new control technologies for soybean black root rot is needed.

[0003] As a biological control technology for soybean black root rot, for example, Patent Document 1 discloses a soybean black root rot control agent containing an active ingredient derived from Bacillus bacteria that produces alkaline substances. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2018-052876 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a novel agent for controlling soybean black root rot fungus. Another object of the present invention is to provide a Calonectria irishicola strain for use in the agent for controlling soybean black root rot fungus. [Means for solving the problem]

[0006] The present inventors discovered that when soybean plants were grown in a culture medium containing a mixture of the soybean black root rot pathogen Calonectria irishicola UH2-1 strain and a novel attenuated Calonectria irishicola UH2-1A strain (Accession Number: NITE-AP-04095), the degree of disease development in the soybean plants was suppressed compared to when the soybean plants were grown in a culture medium containing only the pathogen, leading to the completion of the present invention.

[0007] That is, the present invention provides, for example, the following inventions. [1] A soybean black root rot fungus control agent containing an attenuated Calonectria ilicicola strain as the active ingredient. [2] The agent for controlling soybean black root rot fungus according to [1], wherein the attenuated Calonectria ilicicola strain is a Calonectria ilicicola strain having accession number NITE-AP-04095. [3] Calonectria ilicicola strain, with accession number NITE-AP-04095. [Effects of the Invention]

[0008] According to the present invention, there is provided a novel agent for controlling soybean black root rot fungus. According to the present invention, there is also provided a Calonectria irishicola strain for use in the agent for controlling soybean black root rot fungus. [Brief explanation of the drawings]

[0009] [Figure 1]1 shows images of representative soybean plants in each treatment group in Example 2. [Figure 2] 1 is a graph showing the results of subjective evaluation of the degree of disease development in the roots of soybean plants in each treatment group in Example 2. [Figure 3] 1 is a graph showing the results of subjective evaluation of the degree of disease development in the roots of soybean plants in each treatment group in Example 3. [Figure 4] 1 is a graph showing the results of subjective evaluation of the degree of disease development in the roots of soybean plants in each treatment group in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0011] [Calonectria ilisicola UH2-1A strain] The Calonectria ilicicola UH2-1A strain according to this embodiment is a novel Calonectria ilicicola strain created by the present inventors. The Calonectria ilicicola UH2-1A strain has been deposited at the National Institute of Technology and Evaluation (NPMD) Patent Microorganisms Depositary (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) under the accession number NITE-AP-04095 (accession date: March 4, 2024).

[0012] Calonectria irishicola is known as the pathogenic fungus that causes soybean black root rot. Calonectria irishicola UH2-1 strain is highly pathogenic to soybean black root rot. On the other hand, Calonectria irishicola UH2-1A strain exhibits significantly reduced pathogenicity to soybean black root rot compared to Calonectria irishicola UH2-1. Furthermore, when a mixture of Calonectria irishicola UH2-1A and Calonectria irishicola UH2-1 was inoculated into the roots of soybean plants, the development of soybean black root rot caused by Calonectria irishicola UH2-1 was suppressed. These results suggest that Calonectria irishicola UH2-1A strain can suppress the development of soybean black root rot. Here, "inoculation" means contacting soybean plants, particularly the roots of soybean plants.

[0013] As described in detail in the Examples, the Calonectria irishicola UH2-1A strain was produced by subculturing a colony of the soybean black root rot fungus UH2-1 strain on a rich medium, and has been identified as a mildly virulent soybean black root rot fungus.

[0014] [Soybean black root rot fungus control agent] The soybean black root rot fungus control agent according to this embodiment (hereinafter also simply referred to as "the control agent according to this embodiment") contains an attenuated Calonectria ilicicola strain as an active ingredient. Therefore, the soybean black root rot fungus control agent according to this embodiment has an action to control soybean black root rot fungus on soybean plants. The action to control soybean black root rot fungus can also be said to be an action to suppress the onset of soybean black root rot disease.

[0015] The soybean black root rot fungus strain controlled by the control agent of this embodiment is not particularly limited, and may be, for example, any strain of Calonectria irishicola, and may be a particularly highly virulent Calonectria irishicola strain. For example, Calonectria irishicola strains UH2-1, MAFF102004(S4), MAFF102006(S6), Y11-1b, and SN2-1 are available. Calonectria irishicola strains MAFF102004(S4) and MAFF102006(S6) are available from the National Agriculture and Food Research Organization (NARO) and the National Agrobiological Resources Genebank (https: / / www.gene.affrc.go.jp / about.php), and strains UH2-1, SN2-1, and Y11-1b are available from the National Agriculture and Food Research Organization (NARO). The soybean black root rot fungus strain controlled by the control agent according to this embodiment may also be a naturally occurring Calonectria irishicola strain that has not yet been isolated.

[0016] As used herein, soybean refers to the genus Glycine max (L.) Merr. of the family Fabaceae, including cultivated soybean (Glycine max (L.) Merr. subsp. max) and wild soybean (Glycine max (L.) Merr. subsp. soja (Siebold & Zucc.) H. Ohashi = Glycine soja Siebold & Zucc.).

[0017] As used herein, unless otherwise specified, the term "plant" includes whole plants, plant cells, plant protoplasts, plant callus, or plant parts such as embryos, pollen, ovules, gametes, seeds, leaves, flowers, branches, fruits, stems, roots, anthers, etc.

[0018] As used herein, "attenuated Calonectria irishicola" refers to Calonectria irishicola that causes soybean black root rot to a reduced degree when inoculated into the roots of soybean plants.

[0019] Whether the Calonectria irishicola of interest is an "attenuated Calonectria irishicola," i.e., whether it reduces the severity of black root rot disease when inoculated into the roots of soybean plants, can be determined, for example, by the following method. The Calonectria irishicola of interest is inoculated into the roots of soybean plants at varying inoculum concentrations (0.5% to 4.0% w / v%), and the soybean plants are then cultivated. The severity of black root rot disease (demonstrative evaluation value) in the soybean roots is determined by subjective evaluation according to known literature (e.g., Jiang et al., 2020, Agronomy 10, no. 6: 887). If the inoculum concentration is increased, and indicators of suppression of black root rot disease or reduced severity of black root rot disease are observed, the plant can be determined to be an "attenuated Calonectria irishicola." In a subjective evaluation based on the above literature, if the disease severity is 2.0 or less when the inoculum concentration is 1 w / v%, and if the disease severity is 2.5 or less when the inoculum concentration is 4 w / v%, it can be determined to be attenuated Calonectria irishicola.

[0020] Furthermore, whether or not the Calonectria irishicola of interest is an "attenuated Calonectria irishicola" can be determined, for example, by inoculating the Calonectria irishicola of interest into the roots of a soybean plant, cultivating the plant, and conducting a subjective evaluation according to known literature (e.g., Jiang et al., 2020, Agronomy 10, no. 6: 887). When the degree of disease occurrence is 1% to 50% of the degree of disease occurrence when a similar evaluation is conducted using the Calonectria irishicola UH2-1 strain, the plant can be determined to be an attenuated Calonectria irishicola.

[0021] The attenuated Calonectria irishicola of this embodiment includes both artificially attenuated Calonectria irishicola strains and naturally occurring attenuated Calonectria irishicola strains. The former can be obtained, for example, by subculturing on a rich medium as described in the Examples, or by creating an existing Calonectria irishicola strain through genetic manipulation such as mutation and identifying it as an attenuated strain through the above-mentioned philosophical evaluation. The latter can be obtained by isolating Calonectria irishicola from nature, for example, from the roots of diseased soybean plants in a soybean cultivation field using standard methods, and identifying the isolated strain as an attenuated Calonectria irishicola strain through the above-mentioned philosophical evaluation. The attenuated Calonectria irishicola of this embodiment may be, for example, the above-mentioned Calonectria irishicola UH2-1A strain.

[0022] The control agent of this embodiment may consist solely of attenuated Calonectria irishicola, or may additionally contain carriers, excipients, surfactants, binders, dispersants, thickeners, preservatives, antioxidants, pH adjusters, organic acids, amino acids, salt, colorants, flavorings, etc., or may contain a culture medium in which attenuated Calonectria irishicola has been cultured. The control agent of this embodiment may contain live or killed attenuated Calonectria irishicola cells, preferably live cells. The control agent of this embodiment may also contain a microorganism other than attenuated Calonectria irishicola. The control agent of this embodiment may be a culture of attenuated Calonectria irishicola as is, or may be cells isolated after culture, or a dried product of the culture or cells.

[0023] The amount of attenuated Calonectria irishicola contained in the control agent of this embodiment may be, for example, 0.001% by mass or more, 0.01% by mass or more, 0.1% by mass or more, 1% by mass or more, 5% by mass or more, 10% by mass or more, 20% by mass or more, 30% by mass or more, 50% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, 95% by mass or more, or 99% by mass or more, or may be 100% by mass or less, 99% by mass or less, 95% by mass or less, 90% by mass or less, 80% by mass or less, 70% by mass or less, 50% by mass or less, 30% by mass or less, 20% by mass or less, 10% by mass or less, 5% by mass or less, 1% by mass or less, or 0.1% by mass or less.

[0024] The form of the control agent of this embodiment is not particularly limited and may be, for example, a liquid, solid, gel, paste, etc., and can be appropriately selected depending on the usage situation, etc.

[0025] The method of use of the control agent according to this embodiment is not particularly limited, as long as it is applied by contacting it with the roots of soybean plants. For example, the control agent according to this embodiment may be applied by coating, spraying, or immersing soybean seeds with the control agent according to this embodiment, or by cultivating soybean plants in soil mixed with an effective amount of the control agent according to this embodiment. The control agent according to this embodiment may be applied at any time from the time the soybean seeds are germinating until the soybean plants wither, and may be applied only once or multiple times depending on the desired effect. Since infection with soybean black root rot is confirmed at the time of primary leaf expansion, it is preferable to apply it before that time.

[0026] When the control agent of this embodiment is used by applying the control agent of this embodiment to soybean seeds, for example, the amount of Calonectria iricicola contained in the control agent may be 1 to 100% by mass, for example, 1 to 10%, 10 to 50%, or 50 to 100% by mass, based on the total amount of the control agent of this embodiment, and the entire seeds may be coated with this. Furthermore, when the control agent of this embodiment is used by mixing it with soil, for example, the inoculum concentration of the control agent may be 0.1 g to 10 g, 0.5 g to 5 g, 1 g to 3 g, or 1 g to 2 g per 100 mL of soil using an inoculum grown in a bran medium as the inoculum source.

[0027] [Method for controlling soybean black root rot fungus] The method for controlling soybean black root rot fungus according to this embodiment includes a step of contacting the attenuated Calonectria irishicola with the roots of a soybean plant (contact step 1).

[0028] The attenuated Calonectria irishicola is as described above. The contact step 1 is as described in the method for using the control agent according to this embodiment.

[0029] [Method for producing soybean plants] The method for producing a soybean plant according to this embodiment is a method for producing a soybean plant that is resistant to soybean black root rot fungus, and includes a step of contacting the roots of a soybean plant with attenuated Calonectria irishicola (contact step 2).

[0030] The attenuated Calonectria irishicola is as described above. The contact step 2 is as described in the method for using the control agent according to this embodiment.

[0031] The method for producing a soybean plant according to this embodiment may further include, after the contact step 2, a step of cultivating the soybean plant (cultivation step).

[0032] The cultivation conditions for the soybean plants in the cultivation step may be those normally used by those skilled in the art for cultivating soybean plants, and may be appropriately set depending on the state of the soybean plants, the type of attenuated Calonectria irishicola inoculated, and other factors. The temperature may be, for example, 20°C to 30°C. The relative humidity may be, for example, 30% to 70%. The photoperiod may be a 14-hour light / 10-hour dark cycle. [Example]

[0033] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0034] Example 1: Creation of attenuated strains <1. Cultivation of the soybean root rot fungus> (1-1. Barley grain stock for storage) Sterilized barley grains were mixed with 5mm squares of mycelium from the soybean black root fungus (UH2-1 strain) along with Milli-Q water and cultured at 25°C, 30% relative humidity, and light for two weeks. After confirming that the mycelium had spread throughout the barley grains, the grains were spread evenly on a sterile petri dish and dried at 30°C for two days. The dried barley grains were stored at -80°C as a soybean black root fungus barley grain stock.

[0035] (1-2. Cultivation of the soybean root rot fungus) The barley grain stock prepared in step 1 was placed on potato dextrose agar medium (PDA medium, Difco) and cultured at 25°C, 30% relative humidity, and under light conditions. After 10 to 14 days, the culture was terminated and used as a colony of the soybean root rot fungus.

[0036] <2. Inoculation of soybean root rot fungus> The colony of the soybean black root fungus prepared in 1-2 was inoculated onto PDA medium and cultured at 25°C, 30% relative humidity, and under light conditions for 7 days. This procedure was repeated 10, 20, and 30 times, and the resulting UH2-1 strain was inoculated into soybean (cultivar: Enrei) seeds, and its pathogenicity to soybeans was evaluated by visual inspection. Strains with reduced pathogenicity compared to strains subcultured once were considered attenuated. Specifically, strains with an average visual inspection score of approximately 1.5 for the severity of soybean black root rot in the roots were considered attenuated.

[0037] The fungal strains were inoculated as follows. Soybean seeds (cultivar: Enrei) were conditioned at high humidity for 2 to 14 days at 4°C. Ten 5mm squares of the fungal colony prepared in section 1-2 were inoculated into bran medium (sterilized mixture of 15g wheat bran, 15g vermiculite, and 45ml distilled water) and cultured at 25°C, 30% relative humidity, and in the light. The cultures on the 10th to 14th day of culture were used as inoculum. The inoculum was mixed with sterilized potting soil (JA Nippi horticultural potting soil No. 1) at a concentration of 1g w / v% (1g of inoculum containing bran medium per 100ml of soil) and filled into a 4x4 cell tray. The conditioned soybean seeds were placed with the hilum facing downwards, and the top of the seeded cell tray was covered with approximately 5mm of sterilized Super Mix A (Sakata). The soybeans were irrigated by submerged irrigation, and the severity of soybean black root rot in the roots was assessed by visual evaluation (Jiang et al., 2020, Agronomy 10, no. 6: 887) 10 to 14 days after cultivation.

[0038] In the objective evaluation, "0" indicates no visible symptoms, "1" indicates small brown necrotic spots on the taproot, "2" indicates brown necrotic spots spreading to the entire taproot and lateral roots, "3" indicates that more than 50% of the roots have been lost due to rot and severe brown necrosis is observed near the soil surface, "4" indicates that almost all of the roots have been lost due to rot, and "5" indicates that the seedlings have died. A disease severity score of 2.0 or less was considered to indicate that the onset of soybean black root rot was suppressed, and a score of more than 2.0 was considered to indicate that the onset of soybean black root rot was not suppressed.

[0039] Using this method, Calonectria irishicola strain UH2-1A was isolated.

[0040] Example 2: Evaluation of the disease suppression effect of attenuated mutant strains on soybean black root rot 1 The highly virulent Calonectria irishicola UH2-1 strain and the attenuated Calonectria irishicola UH2-1A strain were used, and these strains were inoculated into soybeans either alone or in combination, as in Example 1. Calonectria irishicola UH2-1 strain was obtained from the National Agriculture and Food Research Organization (NARO) (collected in Hyogo Prefecture) and used for the test. Calonectria irishicola UH2-1 strain was inoculated into bran medium and cultured at 25°C, 30% relative humidity, and under light conditions. The culture was used as the inoculum for Calonectria irishicola UH2-1. An inoculum for Calonectria irishicola UH2-1A was also prepared in a similar manner. Only the inoculum of the virulent strain or only the inoculum of the attenuated strain was mixed into the culture soil so that each strain was 1 w / v%. The mixing ratio of the virulent strain and the attenuated strain inoculum was 1:1, and the total was mixed to 2 w / v%. Soybean seeds were sown in the culture soil and cultivated. Thereafter, the degree of soybean black root rot disease was determined by objective evaluation in the same manner as in Example 1. As a control, the degree of soybean black root rot disease was also determined for soybean seeds sown in culture soil containing no inoculum.

[0041] The results are shown in Figures 1 and 2. In Figure 1, (A) to (D) show images of representative soybean plants grown in uninoculated soil ("uninoculated area"), soil containing only virulent strains ("virulent strain inoculated area"), soil containing both virulent and attenuated strains ("mixed inoculation area"), and soil containing only attenuated strains ("attenuated strain inoculated area"). The white bars in Figure 2 indicate the mean value (n = 3), and the error bars indicate the standard deviation. There was a significant difference at the 5% level between the different inoculation areas in Figure 2.

[0042] As shown in Figure 1, in the virulent strain inoculated area (B), growth of soybeans below ground and above ground was suppressed compared to the uninoculated area (A), and the roots were rotten, showing symptoms of black root rot. In the mixed inoculated area (C), growth suppression of soybeans below ground and above ground and symptoms of root rot were alleviated compared to the virulent strain inoculated area (B). In the attenuated strain inoculated area (D), growth suppression of soybeans below ground and above ground due to black root rot infection was alleviated compared to the virulent strain inoculated area (B) and the mixed inoculated area (C).

[0043] As shown in Figure 2, the disease severity in soybeans was 4.3±0.3 in the highly virulent strain inoculated area, 3.3±0.3 in the mixed inoculated area, and 1.7±0.3 in the attenuated strain inoculated area. The results for the highly virulent strain and mixed inoculated areas showed that inoculating soybean seeds with the attenuated strain suppressed the development of soybean black root rot.

[0044] [Example 3: Evaluation of the pathogenicity of soybean black root rot by attenuated mutants] Only the attenuated strain inoculated plots were prepared, and the pathogenicity of the attenuated strain was evaluated. The degree of soybean black root rot disease was evaluated by holistic observation in the same manner as in Example 2, except that the inoculation concentration of the attenuated strain was changed to 0.5 w / v%, 1.0 w / v%, or 4.0 w / v%. The results are shown in Figure 3. Figure 3 shows the average value for n=3, and the error bars in Figure 3 indicate the standard deviation.

[0045] As shown in Figure 3, when the inoculation concentration of the attenuated strain was changed to 0.5 w / v%, 1.0 w / v%, and 4.0 w / v%, the disease severity ranged from 1.4 to 2.1. At all inoculation concentrations, the incidence of soybean black root rot was suppressed compared to the highly virulent strain (Figure 2), and no significant increase in the disease severity was observed even when the inoculation concentration was increased. This suggests that the attenuated strain is highly safe for soybean plants.

[0046] Example 4: Evaluation of the disease suppression effect of attenuated mutant strains on soybean black root rot 2 In Example 2, it was confirmed that the degree of disease development in the roots of soybean plants was suppressed by mixed inoculation with the UH2-1 strain, which was the basis for the attenuated mutant. The following experiment was conducted to verify whether a similar inhibitory effect was observed with other strains of Calonectria irishicola.

[0047] Strains MAFF102004 (S4) and MAFF102006 (S6) were obtained from the National Agriculture and Food Research Organization (NARO) (https: / / www.gene.affrc.go.jp / about.php), and strains Y11-1b, SN2-1, and UH2-1 were obtained from the National Agriculture and Food Research Organization (NARO). The samples were collected from Gunma, Saitama, Ibaraki, Nagano, and Hyogo prefectures, respectively. The severity of soybean black root rot was assessed in the same manner as in Example 2, except that these five strains were designated as virulent strains and a total of 10 treatment plots, consisting of single inoculations of the five strains and mixed inoculations with attenuated strains, were tested in a randomized block design with three blocks. Statistical analysis was performed using a 2 × 5 two-way randomized block analysis of variance. Factors considered were block, presence or absence of attenuated mutants, number of strains, and interaction. The results are shown in Table 1 and Figure 4.

[0048] [Table 1]

[0049] As shown in Table 1, the interaction (A × B) was not significant, so a test of the main effects was performed. The p-values ​​for the presence or absence of attenuated mutants (A) and the number of strains (B) were significant, at less than 0.1%. The mean values ​​for each treatment group were then estimated using the main effects model (Figure 4). Figure 4 shows that the difference in mean disease severity between the single-inoculation and mixed-inoculation groups was 0.7, with a 95% confidence interval of [0.41, 0.99]. This means that, regardless of the strain, the severity of disease in the roots of the mixed-inoculation group was significantly lower than that of the single-inoculation group.

Claims

1. A soybean black root rot fungus control agent containing an attenuated Calonectoria ilisicola strain as an active ingredient.

2. 2. The soybean black root rot fungus control agent according to claim 1, wherein the attenuated Calonectria ilisicola strain is a Calonectria ilisicola strain having the accession number NITE-AP-04095.

3. Calonectria ilisicola strain, accession number NITE-AP-04095.

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