A novel soybean rhizobia

Abstract

To provide root nodulating bacteria that can survive even in aerobic conditions and are resistant to environmental stress, and a microbial material including the same.SOLUTION: A microbial material is prepared by adding and mixing soybean-root nodulating bacteria (APU-NS64 strain) to and with a material carrying the soybean-root nodulating bacteria (APU-NS64 strain) that have NapA and NosZ and can be grown anaerobically.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to soybean root nodule bacteria, and more particularly to root nodule bacteria that are resistant to environmental stress and can survive under anaerobic conditions. [Background technology]

[0002] Soybeans are an important agricultural product worldwide. The global soybean yield in 2018 was 285 kg / 10 a (USDA, 2019). In contrast, Japan's yield was 167 kg / 10 a, which is lower than that of major soybean-producing countries (Ministry of Agriculture, Forestry and Fisheries, 2019).

[0003] Soybean cropping systems include continuous cropping of soybeans with other field crops, crop rotation with other field crops, and rice paddy-converted fields. In recent years, however, the rice paddy-converted fields system, which is effective in reducing damage from continuous cropping and reducing weeds, has become the mainstream.

[0004] It has been reported that of the 150,000 hectares of soybean cultivation area in Japan in 2017, 80.2%, or 123,000 hectares, was converted to paddy field (Ministry of Agriculture, Forestry and Fisheries, 2019).

[0005] Heavy clay soils in paddy field conversion systems have poor drainage and breathability, which inhibit soybean growth and suppress nodule formation, resulting in reduced yields.

[0006] Soybean is a highly nitrogen-requiring crop, and fixed nitrogen through symbiosis with rhizobia plays an important role as a nitrogen source.

[0007] Rhizobium forms nodules on legume plants, and known species include Rhizobium, Bradyrhizobium, and Azorhizobium. These rhizobium bacteria are commercially available as inoculants and are used in the cultivation of legume crops and legume pastures.

[0008] The applicant of the present application has proposed a hairy vetch rhizobia strain that has high symbiotic nitrogen fixation activity even under low temperature conditions, with regard to the rhizobia of hairy vetch (Vicia villosa Roth), a legume used as green manure (Patent Document 1).

[0009] Because rhizobia are aerobic, if the soil has poor drainage, the rhizobia that have adapted to that soil cannot survive, inhibiting nodule formation on the host. It has also been reported that if the density of rhizobia in the soil is low or if there are few good rhizobia, nodule formation is similarly inhibited, affecting soybean growth and yield.

[0010] Therefore, the technology to artificially inoculate superior rhizobia that are resistant to environmental stress and have a higher priority than indigenous rhizobia is considered important for improving soybean productivity.

[0011] To date, superior rhizobia with high nitrogen fixation ability have been selected and used as microbial materials, but their effectiveness is often unclear due to reasons such as their inability to adapt to soil environments. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] Patent No. 4955431 Summary of the Invention [Problem to be solved by the invention]

[0013] The present invention aims to propose a root nodule bacterium that can survive under anaerobic conditions and is resistant to environmental stress, and a microbial material that utilizes the same. [Means for solving the problem]

[0014] [1] A soybean rhizobia (strain APU-NS64) that possesses NapA and NosZ and is capable of anaerobic growth.

[0015] [2] A microbial material obtained by adding and mixing the soybean rhizobia (APU-NS64 strain) according to claim 1 to a carrier material.

[0016] [3] A microbial material according to [2], which is prepared by adding liquid-cultured soybean rhizobia (APU-NS64 strain) according to claim 1 in a mass ratio of 1% to 50% to the carrier material, and drying the carrier material until the moisture content is 5 to 10%.

[0017] [4] The microbial material according to [2] or [3], wherein the carrier material is a clay mineral.

[0018] [5] The microbial material according to [2] or [3], wherein the carrier material is an organic raw material.

[0019] [6] The microbial material according to [2] or [3], wherein the carrier material is an ash raw material.

[0020] [7] A microbial material comprising a granular or particulate carrier material coated with the soybean rhizobia (APU-NS64 strain) [1].

[0021] [8] The microbial material according to [7], wherein the carrier material is a clay mineral.

[0022] [9] [7] A microbial material according to [7], wherein the carrier material is an organic raw material.

[0023]

[10] A microbial material according to [7], wherein the carrier material is an ash raw material.

[0024]

[11] A fertilizer obtained by adding and mixing the microbial material of [2] or [7] in an amount of 0.5 to 1.0% by mass to a chemical fertilizer or an organic fertilizer.

[0025]

[12] A fertilizer comprising a granular or granular chemical fertilizer or organic fertilizer coated with 0.5 to 1.0 mass% of the microbial material of [2] or [7].

[0026] In the above, examples of clay mineral carriers include zeolite and diatomaceous earth, examples of organic raw material carriers include livestock manure compost and castor meal, and examples of ash raw material carriers include wood ash and rice husk ash. [Effects of the Invention]

[0027] According to this invention, it is possible to provide rhizobia that are resistant to environmental stress and can survive under anaerobic conditions, and a microbial material using the same. According to this invention, it is possible to provide a technique for artificially inoculating superior rhizobia that are resistant to environmental stress and are preferred species over indigenous rhizobia, which can contribute to improving soybean productivity.

[0028] The present invention provides superior rhizobia that can survive in paddy fields and anaerobic soil environments with poor drainage. It also provides a microbial material utilizing such superior rhizobia and a method for producing the material.

[0029] The microbial material prepared using the rhizobia of this invention can stably form nodules even in soybean cultivation in poorly drained environments after conversion from paddy fields, and can contribute to stabilizing yields through efficient symbiotic nitrogen fixation. [Brief explanation of the drawings]

[0030] [Figure 1] Reference photographs illustrating the growth ability of isolated bacterium strain 1604 (APU-NS64 strain) under anaerobic conditions. [Figure 2] A graph showing the growth curve of isolated strain 1604 (APU-NS64 strain). [Figure 3] Reference photograph showing the post-electrophoresis gel for the NapA gene. [Figure 4] Reference photograph showing the post-electrophoresis gel for the NosZ gene. [Figure 5]Diagram showing nitrogen fixation activity per nodule. DETAILED DESCRIPTION OF THE INVENTION

[0031] <Isolation of soybean rhizobia (strain APU-NS64) capable of anaerobic growth> Test Case 1 The bacteria were isolated from soybean root nodules grown in heavy clay soil in a field within the Field Center of the Faculty of Bioresource Sciences, Akita Prefectural University, which had poor drainage and survived even under waterlogged conditions.

[0032] The collected nodules were removed from the roots and sterilized using 70% ethanol and 0.5% sodium hypochlorite in the same manner as for soybean seeds, and then placed in sterilized water.

[0033] The nodules were cut with a scalpel in a clean bench, the infected area inside the nodule was touched with a platinum loop, and the bacteria were inoculated onto a YM plate medium, which was then cultured at 35°C in the dark for 2 weeks under aerobic conditions.

[0034] The detected bacteria were continuously isolated and cultured until they became single colonies.

[0035] <Anaerobic growth ability of isolated strains> To examine the ability of the isolated strains to grow anaerobically, the rhizobia culture solution of the isolated strains grown in YM liquid Thai medium was diluted to 102 cells / mL. The diluted culture solution was inoculated onto YM plates containing bromothymol blue (BTB). The medium was placed in a sealed plastic container with an oxygen absorber (Anelopac Kenki, Sugiyama Gen) and an anaerobic indicator (Anelopac® Anaerobic Indicator, Sugiyama Gen), creating anaerobic conditions, and the strains were incubated at 35°C in the dark.

[0036] After the incubation, the presence or absence of colony formation was visually confirmed, and the anaerobic growth ability of the isolated strains was evaluated.

[0037] As a control, the growth of Bradyrhizobium USDA110 strain was evaluated under the same culture conditions.

[0038] <Detection of nitrate reduction genes from isolated strains> To detect the nitrate reduction genes of the isolated strains, rhizobia DNA was extracted. Total DNA of each rhizobia strain cultured in YM liquid medium was extracted using a DNA extraction kit (Wizard® Genomic DNA Primition Kit: Promega). 1 mL of rhizobia culture was placed in a 1.5 mL tube in a clean bench. The tube was centrifuged at 10,000 × g at 4 °C for 2 minutes, and the supernatant was discarded. 1 mL of sterilized water was added, and the tube was centrifuged at 15,000 × g at 4 °C for 2 minutes, and the supernatant was discarded. 600 μL of Nuclei Lysis Solution was added, and the mixture was stirred using a vortex mixer.

[0039] The mixture was incubated at 80°C for 5 minutes using an aluminum block heater (ALB-221: IWAKI) and then cooled to room temperature. 3 μL of RNase Solution was added and the mixture was stirred upside down several times. The mixture was then incubated in a 37°C water bath for 60 minutes and then cooled to room temperature.

[0040] 200 μL of Protein Precipitation Solution was added, and the mixture was vortexed for 20 seconds, then immediately incubated on ice for 5 minutes. The mixture was centrifuged at 4°C and 15,000 × g for 3 minutes.

[0041] After centrifugation, 500 μL of the supernatant was taken and transferred to a 1.5 mL microtube containing 600 μL of isopropanol solution, and gently stirred up and down.

[0042] After centrifugation at 4°C and 15,000 xg for 2 minutes, the supernatant was removed and 600 µL of 70% ethanol was added, followed by gentle up-and-down stirring.

[0043] After centrifugation at 15,000×g at 4° C. for 2 minutes, the 70% ethanol was removed as much as possible, and the mixture was dried using an evaporator for about an hour until the ethanol was completely dried.

[0044] 100 μL of DNA Rehydration Solution was added to dissolve the DNA, and the mixture was stored frozen at -20°C until use.

[0045] The extracted rhizobia DNA was used as a template for PCR amplification using a primer set targeting the nitrate reduction system genes (napA, nirK, norC, nosZ) of B. diazoefficiens USDA110. The primer sequences are shown in Table 1, and the PCR conditions are shown in Table 2.

[0046] [Table 1]

[0047] [Table 2]

[0048] Electrophoresis was performed on a 2% agarose gel at 100 V for 30 minutes. After electrophoresis, the gel was immersed in a solution containing 200 mL of ion-exchanged water and 10 μL of ethidium bromide for 7 minutes. Bands were detected using an LED illuminator (LB-16BG: Nippon Genetics).

[0049] As a result of the test, the standard strain USDA110 did not grow at all under anaerobic conditions, but growth was confirmed in the isolated strain mentioned above ("Isolated strain (1604)" on the right side of Figure 1) (Figure 1).

[0050] Furthermore, the growth rate of the above-mentioned isolated strains in culture medium under normal conditions (aeration conditions) was faster than that of the standard strain USDA110, demonstrating that they have commercially advantageous characteristics (Figure 2).

[0051] This made it possible to isolate a soybean rhizobia capable of anaerobic growth ("Isolated strain (1604)" on the right side of Figure 1).

[0052] The isolated strain (1604 strains) was deposited at the National Institute of Technology and Evaluation (NITE) as strain APU-NS64, and a certificate of deposit and viability were issued under the accession number NITE p-03443.

[0053] Analysis of the NapA gene post-electrophoresis gel (Fig. 3) and the NosZ gene post-electrophoresis gel (Fig. 4) for the isolated soybean rhizobia capable of anaerobic growth ("Isolate (1604)" on the right side of Fig. 1) showed that the isolated soybean rhizobia capable of anaerobic growth ("Isolate (1604)" on the right side of Fig. 1) possesses the nitrate reductase gene napA and the nitrous oxide enzyme gene nosZ (Table 3, Fig. 3, Fig. 4).

[0054] The control Bradyrhizobium USDA110 strain also possesses the same gene, but it was confirmed that it cannot grow under anaerobic conditions (Table 3).

[0055] [Table 3]

[0056] <Pot cultivation test> The anaerobic strain (1604 strain) isolated as described above and carrying the nitrate reductase gene napA and the nitrous oxide enzyme gene nosZ, and the standard strain USDA110, were inoculated into soybeans and grown in pots under normal and over-humid conditions (high groundwater level).

[0057] Thirty days after sowing, nitrogen fixation activity (acetylene reduction activity) was measured.

[0058] Under normal conditions, the 1604 strain inoculated plot showed higher nitrogen fixation activity than the USDA110 strain inoculated plot, and this was particularly evident under excessively wet conditions (Fig. 5).

[0059] Therefore, it was revealed that the anaerobically growing strain (strain 1604) isolated as described above and possessing the nitrate reductase gene napA and the nitrous oxide enzyme gene nosZ is an excellent strain that can maintain high nitrogen fixation activity even under conditions of excessive soil moisture.

[0060] <Preparation of rhizobia inoculation material> A rhizobial inoculant was prepared using the anaerobic strain (strain 1604) isolated as described above and carrying the nitrate reductase gene napA and the nitrous oxide enzyme gene nosZ.

[0061] The isolated rhizobia strain 1604 was cultured in YM liquid medium for 7 days and then adjusted to a concentration of 1 x 108 colony forming unit (cfu) / ml.

[0062] It was added to powdered zeolite at a weight ratio of 50%, and the moisture content of the final material was adjusted to 5-10% using a forced-air dryer.

[0063] As a result, it became possible to prepare rhizobia inoculation materials using anaerobic strains (1604 strains) isolated as described above that possess the nitrate reductase gene napA and the nitrous oxide enzyme gene nosZ, including rhizobia strains (1604 strains) with a cfu / g or higher.

[0064] The prepared rhizobia inoculant was usable by adding it to chemical or organic fertilizers at a weight ratio of 0.5-1.0%, for example, by coating granular chemical or organic fertilizers with it at a weight ratio of 0.5-1.0%.

[0065] <Application test (Hokkaido)> An application test on soybeans was conducted in Wassamu Town, Hokkaido in 2019. The test plants were soybeans (Glycine max), and the variety was Yukihomare.

[0066] The test scale was 15a for each plot, and chemical fertilizer was used in the control plot, while organic chemical fertilizer (NPK = 9-20-13) coated with the rhizobia material prepared as described above was used in the test plot. Both fertilizers were applied to the side rows at 2-3 kgN / 10a.

[0067] Soybeans were sown in mid-May at a seeding rate of 9 kg / 10 a. A yield survey was conducted in mid-October to evaluate the effectiveness of the materials. The results of the test showed that the total number of pods increased in the test plots compared to the control plots, and the yield tended to increase (Table 4).

[0068] [Table 4]

[0069] <Application test (Akita Prefecture)> Test field: Akita Prefectural University Field Education and Research Center, converted paddy field (field history: continuous paddy field until 2011, soybean cultivation in 2012, and since then, paddy field and soybean cultivation alternated every year) Test crop: Soybean [Glycine max (L.) Merr.] (variety: Ryuho) Cultivation management: The field was plowed on June 19, 2020, and seeds were sown on the same day with a furrow spacing of 72 cm and a plant spacing of 19 cm, with two seeds.

[0070] In the control plot, chemical fertilizer was applied at the side dressing of 4 kg / 10 a (N-2. kg / 10 a) at the time of sowing.

[0071] In the test plot (plot inoculated with 1604 strains), the rhizobia material prepared as described above was mixed with chemical fertilizer at a ratio of 1% by weight and applied as side dressing at the time of sowing.

[0072] The soil was cultivated in early July and the crop was harvested on October 22nd.

[0073] In the test area (inoculated with 1604 strain), infection with the inoculated rhizobia strain 1604 was confirmed in approximately 10% of the nodules. The infection rate with seed dressing, a common inoculation method, has been reported to be only a few percent, so this inoculation method was considered to be more effective than conventional inoculation methods.

[0074] The yield in the test area (inoculated with 1604 strains) tended to be higher than in the control area (Table 5). This suggests that the amount of photosynthetic products consumed by the root nodules in the test area (inoculated with 1604 strains) was less than in the control area, and that the resulting photosynthetic products were used for the growth of other organs, resulting in an increase in the number of pods and a higher yield than in the control area.

[0075] [Table 5]

Claims

1. A soybean rhizobia (strain APU-NS64) that possesses NapA and NosZ and is capable of anaerobic growth.

2. A microbial material comprising a carrier material to which the soybean rhizobia (APU-NS64 strain) according to claim 1 is added and mixed.

3. The microbial material according to claim 2, wherein the liquid-cultured soybean rhizobia (APU-NS64 strain) according to claim 1 is added to the carrier material in a mass ratio of 1% to 50%, and the carrier material is dried to a moisture content of 5 to 10%.

4. 4. The microbial material according to claim 2, wherein the carrier material is a clay mineral.

5. 4. The microbial material according to claim 2, wherein the carrier material is an organic raw material.

6. 4. The microbial material according to claim 2, wherein the carrier material is an ash material.

7. A microbial material comprising a granular or particulate carrier material coated with the soybean rhizobia (APU-NS64 strain) according to claim 1.

8. 8. The microbial material according to claim 7, wherein the carrier material is a clay mineral.

9. 8. The microbial material according to claim 7, wherein the carrier material is an organic raw material.

10. 8. The microbial material according to claim 7, wherein said carrier material is an ash material.

11. A fertilizer obtained by adding and mixing the microbial material according to claim 2 or claim 7 in an amount of 0.5 to 1.0% by mass to a chemical fertilizer or an organic fertilizer.

12. A fertilizer comprising a granular or granular chemical fertilizer or organic fertilizer coated with the microbial material according to claim 2 or claim 7 in an amount of 0.5 to 1.0% by mass.

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