Soybean soil-borne disease mitigation agent

Abstract

To provide novel microorganisms having antagonistic action to pathogenic microorganisms of soil-borne soybean diseases and uses thereof.SOLUTION: Disclosed are two filamentous fungi (Accession Number: NITE P-03445 and NITE P-03488) having antagonistic actions to pathogenic microorganisms that cause soil-borne soybean diseases. Also disclosed is a soil-borne soybean disease-mitigation material comprising the fungi (NITE P-03445 and / or NITE P-03488) as an effective ingredient, or comprising substances derived from the fungi (NITE P-03445 and / or NITE P-03488) as an effective ingredient.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to novel microorganisms and methods for using them. In particular, it relates to novel microorganisms having an antagonistic effect against soybean soil-borne pathogenic bacteria and methods for using them.

Background Art

[0002] Soybeans are important agricultural products worldwide. The world soybean yield in 2018 was 285 kg / 10a (USDA, 2019). In contrast, the domestic yield is 167 kg / 10a, which is lower compared to major producing countries (Ministry of Agriculture, Forestry and Fisheries, 2019).

[0003] The reasons for the low domestic soybean yield are considered to include waterlogging due to poor soil drainage, the influence of paddy field conversion fields, continuous cropping obstacles due to increased planting frequency, and frequent occurrence of diseases.

[0004] In particular, as soil diseases, the occurrence of soil diseases such as black root rot and soybean stem blight has a great impact, but a complete control method has not been established, and it is in a situation that has become a major problem.

[0005] Soybean black root rot is a soil disease caused by the soybean black root rot fungus (scientific name: Calonectria ilicicola), which is a type of fungus. After the flowering stage, chlorotic lesions can be seen on the leaves, and red perithecia are formed at the ground level (Non-Patent Document 1, Non-Patent Document 2).

[0006] Non-Patent Document 3 shows that the soybean black root rot fungus can survive and infect in the soil in the form of microsclerotia for at least 7 years even in an environment where the host plant is not planted, suggesting that in order to prevent the occurrence of soybean black root rot, it is necessary to suppress the growth of the soybean black root rot fungus in the soil or reduce the fungal density.

[0007] Soybean stem blight is a disease caused by a type of oomycete called Phytophthora sojae. It forms zoosporangia from oospores or diseased tissue, releasing numerous zoospores that spread infection and damage. Since the zoospores remain in the soil for a long time and reach the host via water, improving soil drainage is considered one of the important control techniques.

[0008] Preventing the occurrence of such soilborne diseases requires many tasks, including improving soil drainage and supplying organic matter.

[0009] Furthermore, in soybean cultivation, the roots are cut during intertillage, leading to continuous infection by pathogens, making complete control difficult.

[0010] The applicant has proposed a soybean black root rot control agent, a microbial material that suppresses soybean black root rot, a soybean black root rot control method, and a soil-borne disease mitigation material that has an inhibitory effect on the growth of soybean black root rot fungi, utilizing the Bacillus bacteria strains APU-W01 (NITE P-02337), APU-O02 (NITE P-02338), and APU-T03 (NITE P-02354) (Patent Document 1, Patent Document 2). [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] Patent No. 6824519 [Patent Document 2] Japanese Patent Publication No. 2019-147757 [Non-patent literature]

[0012] [Non-Patent Document 1] Nishi, Abstracts of the Japan Society of Crop Science Conference, 225, 346-347 (2008) [Non-Patent Document 2] Ochi, Hokkaido University, Doctoral Dissertation, No. 6913 (2014) [Non-Patent Document 3] Nishi, Sato, Annual Report of the Kanto-Tosan Plant Protection Society, 41, 45-46 (1994) [Overview of the project] [Problems that the invention aims to solve]

[0013] The soil-borne disease mitigation materials proposed in Patent Documents 1 and 2 contain Bacillus bacteria that are present on the root surface and exert an antagonistic effect against black root rot fungi through the enzymatic activity of chitinases and other enzymes they possess.

[0014] On the other hand, the inventors of this invention have found that soybean stem blight fungus cannot be controlled solely by the action of Bacllus bacteria proposed in Patent Documents 1 and 2.

[0015] This patent application aims to provide a novel microorganism that has antagonistic activity against soybean soil-borne pathogens and to offer a way to utilize it. [Means for solving the problem]

[0016] The present invention, which solves the above problems, can be exemplified as follows. [1] Filamentous fungi under accession number NITE P-03445 that have antagonistic effects against soybean soil-borne pathogens, and filamentous fungi under accession number NITE P-03488 that have antagonistic effects against soybean soil-borne pathogens.

[0017] [2] It contains as an active ingredient a filamentous fungus that has antagonistic activity against soybean soil-borne pathogens and is authorized under accession number NITE P-03445, and / or it contains as an active ingredient a filamentous fungus that has antagonistic activity against soybean soil-borne pathogens and is authorized under accession number NITE P-03488, or, A substance derived from a filamentous fungus deposited under accession number NITE P-03445, which has an antagonistic effect against soybean soil-borne pathogenic fungi, and / or a substance derived from a filamentous fungus deposited under accession number NITE P-03488, which has an antagonistic effect against soybean soil-borne pathogenic fungi, are included as active ingredients Soybean soil-borne disease reducing material.

[0018] [3] A filamentous fungus deposited under accession number NITE P-03445, which has an antagonistic effect against soybean soil-borne pathogenic fungi, and / or a filamentous fungus deposited under accession number NITE P-03488, which has an antagonistic effect against soybean soil-borne pathogenic fungi, or A substance derived from a filamentous fungus deposited under accession number NITE P-03445, which has an antagonistic effect against soybean soil-borne pathogenic fungi, and / or a substance derived from a filamentous fungus deposited under accession number NITE P-03488, which has an antagonistic effect against soybean soil-borne pathogenic fungi A soybean soil-borne disease reducing material obtained by adding and mixing the above to a carrier.

[0019] [4] A filamentous fungus deposited under accession number NITE P-03445, which has an antagonistic effect against liquid-cultured soybean soil-borne pathogenic fungi, and / or a filamentous fungus deposited under accession number NITE P-03488, which has an antagonistic effect against liquid-cultured soybean soil-borne pathogenic fungi, or A liquid substance derived from a filamentous fungus deposited under accession number NITE P-03445, which has an antagonistic effect against soybean soil-borne pathogenic fungi, and / or a liquid substance derived from a filamentous fungus deposited under accession number NITE P-03488, which has an antagonistic effect against soybean soil-borne pathogenic fungi A soybean soil-borne disease reducing material obtained by adding 1% - 50% by mass ratio of the above to a carrier and drying until the moisture content becomes 5 - 10%.

[0020] [5] The soybean soil-borne disease reducing material as described in [4], where the carrier is a clay mineral.

[0021] [6] A soybean soil disease mitigation material wherein the carrier is made from organic raw materials [4].

[0022] [7] A soybean soil disease mitigation material wherein the carrier is made from ash [4].

[0023] [8] A Bacillus-containing material is made by incorporating three types of Bacillus bacteria with different characteristics into a carrier material: Bacillus amyloliquefaciens (accession number: NITE P-02337), Bacillus subtilis (accession number: NITE P-02338), and Bacillus subtilis (accession number: NITE P-02354). A soybean soil-borne disease mitigation material obtained by adding and mixing any of the soybean soil-borne disease mitigation materials [2] to [7].

[0024] [9] A soybean soil disease mitigating material [8] obtained by adding and mixing the soybean soil disease mitigating material described in any one of claims 2 to 7 in a mass ratio of 0.5 to 1.0% to the Bacillus bacteria-containing material.

[0025]

[10] A granular or granular Bacillus bacteria-containing material is made by incorporating three types of Bacillus bacteria with different characteristics, namely Bacillus amyloliquefaciens (accession number: NITE P-02337), Bacillus subtilis (accession number: NITE P-02338), and Bacillus subtilis (accession number: NITE P-02354), into a carrier material. A soybean soil-borne disease mitigating material comprising a coating of any of the soybean soil-borne disease mitigating materials [2] to [7].

[0026]

[11] In the granular or granular Bacillus bacteria-containing material A soybean soil disease mitigating material

[10] comprising coating with one of the soybean soil disease mitigating materials [2] to [5] in a mass ratio of 0.5 to 1.0%.

[0027] As a Bacillus-containing material, which includes three types of Bacillus bacteria with different characteristics, namely Bacillus amyloliquefaciens (accession number: NITE P-02337), Bacillus subtilis (accession number: NITE P-02338), and Bacillus subtilis (accession number: NITE P-02354), in the carrier material described above, or as a granular or granular Bacillus-containing material, the one proposed by the present applicant in a previous patent application (Japanese Patent Publication No. 2019-147757, Soilborne Disease Mitigation Material) can be used.

[0028] The filamentous fungi under accession number NITE P-03445, which have antagonistic effects against soybean soil-borne pathogens, and the filamentous fungi under accession number NITE P-03488, which also have antagonistic effects against soybean soil-borne pathogens, can exert the desired effects when used individually, but using them simultaneously can produce a more stable effect.

[0029] In other words, by using two types of filamentous fungi with different characteristics, the mitigating effect against soybean stem blight is stabilized.

[0030] As described above, by encapsulating two types of filamentous fungi with different characteristics in a carrier, the mitigating effect against soybean stem blight is stabilized. [Effects of the Invention]

[0031] This invention provides a novel microorganism that has antagonistic activity against soybean soil-borne pathogens and its use. [Brief explanation of the drawing]

[0032] [Figure 1] Reference photograph showing the results of a confrontation culture between the isolated strain and the soybean black root rot fungus. [Figure 2] Reference photograph showing the results of confrontation culture between isolated strains and soybean stem blight. [Figure 3] A reference photo showing the results of a pot cultivation experiment. [Figure 4] Reference photograph illustrating the process of mycelial formation from materials. [Figure 5] Changes in soil microbial flora due to application of materials containing Bacillus bacteria and materials containing Bacillus bacteria and Cunninghamella elagans (reference photo showing the results of confirming the number of bands by electrophoresis). [Figure 6] This figure shows the results of the evaluation of the effects of a material containing the Bacillus genus bacterium Cunninghamella elagans on stem blight pathogens in soil using the ARISA method. [Modes for carrying out the invention]

[0033] <Isolation of a bacterium that has antagonistic effects against soybean soil-borne pathogens> Two types of filamentous fungi were isolated from soil in Niigata City, Niigata Prefecture.

[0034] The isolated filamentous fungi were subjected to DNA extraction, the ITS gene region was amplified by PCR, and the base sequence was determined by DNA sequencing analysis at the Akita Prefectural University Research Support Center for Life Sciences to identify the isolated filamentous fungi. The identification results showed that the fungi were two species: Cunninghamella elagans (APU-YM-01 strain) and Aspergillus aureoles (APU-YM-02 strain).

[0035] Cunninghamella elagans (APU-YM-01 strain) and Aspergillus aureoles (APU-YM-02 strain) have been deposited with the National Institute of Technology and Evaluation (NITE). Cunninghamella elagans (APU-YM-01 strain) is deposited under accession number NITE P-03488, and Aspergillus aureoles (APU-YM-02 strain) is deposited under accession number NITE P-03445.

[0036] <Confirmation of antagonistic effects against soybean black root rot fungus and stem blight fungus> As described above, the antagonistic activity of the isolated strains against soybean black root rot fungus and stem blight fungus was tested.

[0037] For soybean black root rot, we used strain AP-12 isolated from a field with a high incidence of disease in Akita Prefecture. For soybean stem blight, we used strains 305,923 and 235,803 distributed by MAFF.

[0038] Two strains were isolated: Cunninghamella elagans (APU-YM-01 strain) (sometimes referred to as "strain R") and Aspergillus aureoles (APU-YM-02 strain) (sometimes referred to as "strain W").

[0039] The antagonistic effect of soybean black root rot fungus was evaluated by placing isolated strains and the causative fungus side by side on PDA medium and assessing the inhibitory effect.

[0040] The antagonistic effect of the soybean stem blight fungus was evaluated by arranging the isolated strain and the causative fungus on CMA medium in the same manner as described above, and assessing the inhibitory effect.

[0041] The culture was performed using a constant-temperature incubator at 25°C for one week.

[0042] In addition, as a control, the antagonistic effect of Bacillus amyloliquefaciens (strain O) against stem blight fungus was compared.

[0043] Figure 1 shows the results of confrontation cultures of soybean black root rot fungus and isolated strains.

[0044] The test results showed that strain R, although not forming a confrontation surface, proliferated to cover the pathogen, physically suppressing its growth. Strain W formed a confrontation surface and suppressed the growth of the pathogen by producing antibiotics and repellents.

[0045] The isolated strains Cunninghamella elagans (APU-YM-01 strain) (R strain) and Aspergillus aureoles (APU-YM-02 strain) (W strain) were both confirmed to have antagonistic activity against soybean black root rot fungus.

[0046] Figure 2 shows the results of confrontation cultures of soybean stem blight fungus and isolated strains.

[0047] The test results showed that strain R spread its hyphae throughout the entire petri dish, forming an opposing surface and exhibiting antagonistic activity against both strains 305923 and 235803 of the soybean stem blight fungus. Strain W grew more slowly than strain R, but also formed an opposing surface and exhibited antagonistic activity against both strains 305923 and 235803 of the soybean stem blight fungus.

[0048] On the other hand, the control strain O formed an antagonistic relationship with soybean stem blight fungus strain 305923, demonstrating antagonistic activity. However, no antagonistic relationship was formed with strain 235803, and its antagonistic activity was not clearly evident.

[0049] The isolated strains Cunninghamella elagans (APU-YM-01 strain) (R strain) and Aspergillus aureoles (APU-YM-02 strain) (W strain) were both confirmed to have antagonistic activity against soybean stem blight fungus.

[0050] <Pot cultivation trial> A culture medium of vermiculite and sand (1:1 ratio) was inoculated with a large amount of soybean black root rot fungus (AP-12 strain) to prepare the material.

[0051] As described above, a material was prepared by inoculating a large amount of soybean black root rot fungus (AP-12 strain) into a culture medium of vermiculite + sand (1:1), and then mixing in strains R (Cunninghamella elagans) and W (Aspergillus aureoles) to prepare the material for the example.

[0052] Materials prepared by inoculating a large amount of soybean black root rot pathogen (AP-12 strain) into vermiculite + sand (1:1) culture soil (i.e., materials without filamentous fungus mixture) were applied to the culture soil at an equivalent amount of 200 kg / 10a. As a control plot, soybeans (cultivar Ryuhou) were sown.

[0053] The materials prepared as described above and serving as examples were also applied to the culture soil at an equivalent amount of 200 kg / 10a. As the R+W mixed plot, soybeans (cultivar Ryuhou) were sown.

[0054] The control plot and the R+W mixed plot were each cultivated in an artificial weather chamber at 25°C for 16 hours during the day and 18°C for 8 hours at night for 2 weeks.

[0055] Pot cultivation test results In the control plot, germination was strongly inhibited by the black root rot pathogen, and almost none of them germinated.

[0056] On the other hand, in the R+W mixed plot, germinating individuals were observed, and they grew normally thereafter (Figure 3).

[0057] Therefore, it was suggested that mixing the R strain (Cunninghamella elagans) and the W strain (Aspergillus aureoles) into the materials might reduce the incidence of soybean black root rot, which is a type of soil-borne pathogen.

[0058] <Test of applying materials containing Bacillus bacteria and materials containing Bacillus bacteria and Cunninghamella elagans (field)> The test was carried out in two fields in Tanba Sasayama City, Hyogo Prefecture, where stem blight and black root rot frequently occur.

[0059] The test plants were black soybeans (Glycine max), and Tambakuro was used as the cultivar.

[0060] As a control plot, a plot with fermented chicken manure was set, and as test plots, a plot with materials containing Bacillus bacteria and a plot with materials containing Bacillus bacteria and Cunninghamella elagans were set.

[0061] <Material adjustment> Preparation of materials containing Bacillus bacteria The preparation of the Bacillus bacteria-containing material described in Patent Document 2 involves using chicken manure compost, zeolite, rice bran, and gypsum as raw materials. Three strains of Bacillus bacteria (APU-W01 strain (NITE P-02337), APU-O02 strain (NITE P-02338), and APU-T03 strain (NITE P-02354)) are cultured during the mixing of the raw materials, and then added to the mixture at a concentration of 5.0 × 10⁷ colony forming units (cfu) / ml and a weight ratio of 2.5%.

[0062] After addition, the product was extruded and dried in a kiln at 240°C for 1 hour to adjust the final moisture content to 5-10%.

[0063] The prepared material was adjusted to contain 5.0 × 10⁷ cfu / g of Bacillus bacteria (APU-W01 strain (NITE P-02337), APU-O02 strain (NITE P-02338), and APU-T03 strain (NITE P-02354)).

[0064] Preparation of Bacillus genus Cunninghamella elagans input material The Bacillus bacteria / Cunninghamella elagans-containing material was prepared as described above, then Cunninghamella elagans was cultured in the prepared material, and finally the material mixed with finely powdered zeolite was coated at a weight ratio of 0.5-1.0%.

[0065] The prepared Bacillus bacteria / Cunninghamella elagans sample was adjusted to contain 5.0 × 10⁷ cfu / g of Bacillus strains (APU-W01 (NITE P-02337), APU-O02 (NITE P-02338), and APU-T03 (NITE P-02354)).

[0066] Since the concentration of filamentous fungi could not be measured, after preparing the material containing Bacillus bacteria and Cunninghamella elagans as described above, the moisture content of the material was adjusted to 40%, and the presence of filamentous fungi was confirmed by a culture test at 35°C (Figure 4).

[0067] <Field cultivation trial> The control plot was 8a and the test plots were 17a each. Chicken manure compost in the control plot was applied to the entire layer at a rate of 100kg / 10a, while each of the test materials in the test plots was applied as a side layer at a rate of 100kg / 10a.

[0068] In addition, the same amount of compound fertilizer was applied to each trial. The control group received 6.0 kgN / 10a, 10.6 kg P2O5 / 10a, and 7.0 kg K2O, while the experimental groups received 6.6 kgN / 10a, 8.7 kg P2O5 / 10a, and 6.7 kg K2O. The planting density was 1,389 plants / 10a, with a row spacing of 160 cm and a plant spacing of 45 cm.

[0069] Sowing took place in mid-May, and the marketable yield of edamame was surveyed on October 15th. Marketable yield was measured by selecting 6 plants with moderate growth from 12 surveyed plants at each fixed point. In addition, the soil microbial community at the time of the yield survey was analyzed using the ARISA method.

[0070] <Field cultivation trial results> A comparison of yields between chicken manure compost, a material containing Bacillus bacteria, and a material containing Bacillus bacteria and Cunninghamella elagans revealed that the material containing Bacillus bacteria and Cunninghamella elagans yielded the highest yield, followed by the Bacillus bacteria-containing material, and the chicken manure compost yielded the lowest. The application of each test material increased the number and weight of fertile pods, demonstrating its contribution to increased yield (Tables 1 and 2).

[0071] The changes in the soil microbial flora (confirmation of band numbers by electrophoresis) following the application of materials containing Bacillus species and materials containing Bacillus species and Cunninghamella elagans are shown in the reference photograph in Figure 5. Furthermore, the results of the evaluation of the effect of materials containing Bacillus species and Cunninghamella elagans on stem blight pathogens in the soil using the ARISA method are shown in Figure 6.

[0072] The application of a material containing the bacterium Cunninghamella elagans resulted in an improvement in the diversity of the soil microbial community (Figure 6).

[0073] Furthermore, similar to the results of the confrontation culture in the "Confirmation of Antagonistic Effects on Soybean Black Root Rot and Stem Late Virus" described above, the absence of detection of stem late virus in the test plots treated with Cunninghamella elagans demonstrated that this filamentous fungus has a mitigating effect on soybean stem late disease.

[0074] [Table 1]

[0075] [Table 2]

Claims

1. A filamentous fungus that has antagonistic activity against soybean black root rot fungus and soybean stem blight fungus, which is registered under accession number NITE P-03445, or a filamentous fungus that has antagonistic activity against soybean black root rot fungus and soybean stem blight fungus, which is registered under accession number NITE P-03488.

2. A filamentous fungus, which has antagonistic activity against soybean black root rot fungus and soybean stem blight fungus, is included as an active ingredient under accession number NITE P-03445 and / or a filamentous fungus, which has antagonistic activity against soybean black root rot fungus and soybean stem blight fungus, is included as an active ingredient under accession number NITE P-03488, or It contains as an active ingredient a substance derived from a filamentous fungus that has antagonistic effects against soybean black root rot fungus and soybean stem blight fungus, which is authorized under accession number NITE P-03445, and / or contains as an active ingredient a substance derived from a filamentous fungus that has antagonistic effects against soybean black root rot fungus and soybean stem blight fungus, which is authorized under accession number NITE P-03488. A material used to reduce soil-borne diseases in soybeans.

3. A filamentous fungus under accession number NITE P-03445 that has antagonistic effects against soybean black root rot fungus and soybean stem blight fungus, and / or a filamentous fungus under accession number NITE P-03488 that has antagonistic effects against soybean black root rot fungus and soybean stem blight fungus, or Substances derived from filamentous fungi under accession number NITE P-03445 that have antagonistic effects against soybean black root rot fungus and soybean stem blight fungus, and / or substances derived from filamentous fungi under accession number NITE P-03488 that have antagonistic effects against soybean black root rot fungus and soybean stem blight fungus. A soybean soil-borne disease mitigation material consisting of a carrier and a material.

4. Filamentous fungi under accession number NITE P-03445 that have antagonistic effects against liquid-cultured soybean black root rot fungi and soybean stem blight fungi, and / or filamentous fungi under accession number NITE P-03488 that have antagonistic effects against liquid-cultured soybean black root rot fungi and soybean stem blight fungi, or A liquid substance derived from a filamentous fungus under accession number NITE P-03445 that has antagonistic effects against soybean black root rot fungus and soybean stem blight fungus, and / or a liquid substance derived from a filamentous fungus under accession number NITE P-03488 that has antagonistic effects against soybean black root rot fungus and soybean stem blight fungus. A soybean soil-borne disease mitigation material comprising a carrier and a filamentous fungus or liquid substance contained therein in a mass ratio of 1% to 50% and a moisture content of 5% to 10%.

5. The soybean soil infectious disease mitigation material according to claim 4, wherein the carrier is a clay mineral.

6. The soybean soil disease mitigation material according to claim 4, wherein the carrier is an organic raw material.

7. The soybean soil disease mitigation material according to claim 4, wherein the carrier is made from ash.

8. In a Bacillus-containing material, three types of Bacillus bacteria with different characteristics are included: Bacillus amyloliquefaciens (accession number: NITE P-02337), Bacillus subtilis (accession number: NITE P-02338), and Bacillus subtilis (accession number: NITE P-02354). A soybean soil-borne disease mitigating material containing the soybean soil-borne disease mitigating material described in any one of claims 2 to 7.

9. The soybean soil infectious disease mitigating material according to claim 8, wherein the Bacillus bacteria-containing material contains 0.5 to 1.0% by mass of the soybean soil infectious disease mitigating material according to any one of claims 2 to 7.

10. Granular or granular Bacillus bacteria-containing material containing three types of Bacillus bacteria with different characteristics, namely Bacillus amyloliquefaciens (accession number: NITE P-02337), Bacillus subtilis (accession number: NITE P-02338), and Bacillus subtilis (accession number: NITE P-02354), is provided as a carrier material. A soybean soil-borne disease mitigating material coated with the soybean soil-borne disease mitigating material described in any one of claims 2 to 7.

11. The granular or granular Bacillus bacteria-containing material The soybean soil infectious disease mitigating material according to claim 10, which is coated with the soybean soil infectious disease mitigating material according to any one of claims 2 to 4, wherein the mass ratio of the soybean soil infectious disease mitigating material is 0.5 to 1.0%.

Images