Agrobacterium bacteria, Agrobacterium bacterial strains, Rhizobium bacterial strains, soybean cultivation treatment agents, soybean cultivation soil production method, soybean cultivation seed production method, and soybean cultivation method

Abstract

To provide novel bacteria with the action of mitigating water immersion stress in soybeans.SOLUTION: An Agrobacterium bacterium has a genome DNA with an ANI value of 96% or higher in comparison to the base sequence of the genome DNA identified in a specific sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to novel Agrobacterium bacteria, Agrobacterium bacterial strains, Rhizobium bacterial strains, a treating agent for soybean cultivation, a method for producing soil for soybean cultivation, a method for producing soybean seeds for cultivation, and a method for cultivating soybeans. [Background technology]

[0002] More than 80% of soybean cultivation in Japan is done in fields converted from paddy fields. In many areas, except Hokkaido, the soybean sowing and early growth period overlap with the rainy season. If fields are flooded or submerged during the germination or early growth period of soybeans, the emergence rate of soybeans decreases and early growth is likely to be poor. As a result, soybeans are frequently damaged by water in many areas of Japan. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] James, et al., Flooding-tolerant legume symbioses from the Brazilian Pantanal, New Phytologist (2001) 150:723-738 Summary of the Invention [Problem to be solved by the invention]

[0004] In soybean cultivation, countermeasures such as tillage and fertilization methods, as well as the introduction of disease-resistant strains, have been attempted. However, compared to Hokkaido, where the frequency of water damage is low, other prefectures have yields that are approximately 100 kg lower per 10 ares on average. Soybeans are an essential crop for the Japanese people, but the self-sufficiency rate is only about 8%, so there is a need to improve domestic soybean production.

[0005] There have been reports of symbiotic microorganisms in legumes in Brazil that are tolerant to flooding (Non-Patent Document 1). However, no microorganisms have been identified that are effective against flooding stress during germination and early growth in soybean cultivation.

[0006] An object of the present invention is to provide a novel bacterium that has the effect of alleviating flooding stress in soybeans. [Means for solving the problem]

[0007] One aspect of the present invention is an Agrobacterium bacterium having genomic DNA with an ANI value of 96% or more relative to the nucleotide sequence of the genomic DNA shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0008] One aspect of the present invention is an Agrobacterium sp. strain SSB39 (accession number NITE P-03789), an Agrobacterium sp. strain SSB64 (accession number NITE P-03790), an Agrobacterium sp. strain SSB74 (accession number NITE P-03791), an Agrobacterium sp. strain SSB16 (accession number NITE AP-04005), an Agrobacterium sp. strain SSB19 (accession number NITE AP-04042), a Rhizobium sp. strain SSB22 (accession number NITE AP-04007), a Rhizobium sp. strain SSB50 (accession number NITE AP-04043), or an Agrobacterium sp. strain SSB62 (accession number NITE AP-04009).

[0009] One aspect of the present invention is a treating agent for soybean cultivation, which comprises at least one of the above-mentioned bacteria.

[0010] One aspect of the present invention is a method for producing soil for growing soybeans, which comprises adding at least one species of the above-described bacteria to soil.

[0011] One aspect of the present invention is a method for producing soybean seeds for cultivation, which comprises attaching and / or harboring at least one of the above bacteria to soybean seeds.

[0012] One aspect of the present invention is a method for cultivating soybeans, which comprises applying at least one of the above-described bacteria to a cultivation field, soybean seeds, or soybean plants. [Effects of the Invention]

[0013] The present invention provides a novel bacterium that has the effect of alleviating flooding stress in soybeans. [Brief explanation of the drawings]

[0014] [Figure 1] 1 is a graph showing the above-ground part length of soybeans 7 days after sowing. [Figure 2] 1 is a graph showing the root length of soybeans 7 days after sowing. [Figure 3] 1 is a graph showing the germination rate. [Figure 4] 1 is a graph showing the above-ground part length of soybeans 7 days after sowing. [Figure 5] 1 is a graph showing the root length of soybeans 7 days after sowing. DETAILED DESCRIPTION OF THE INVENTION

[0015] Preferred embodiments of the present invention will be described in detail below, but the present invention is not limited to the following embodiments.

[0016] (bacteria) One embodiment of the present invention is an Agrobacterium bacterium having genomic DNA with an ANI value of 96% or more relative to the genomic DNA base sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 represent the genomic DNA base sequences of the Agrobacterium genus SSB39 strain, Agrobacterium genus SSB64 strain, and Agrobacterium genus SSB74 strain, respectively.

[0017] The Agrobacterium strains SSB39, SSB64, and SSB74 have all been deposited at the National Institute of Technology and Evaluation (NPMD) Patent Microorganisms Deposit Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818), with accession numbers NITE P-03789, NITE P-03790, and NITE P-03791, respectively (deposit date for all strains: December 7, 2022).

[0018] Genome analysis confirmed that the Agrobacterium strains SSB39, SSB64, and SSB74 belong to the genus Agrobacterium, but are not part of any known species within the genus, and are therefore a new species of bacteria. Genomic DNA is considered to be of the same species when its ANI (Average Nucleotide Identity) value is 96% or higher. The Agrobacterium strains SSB39 and SSB74 are considered to be of the same species, as their ANI values ​​are 96% or higher. On the other hand, the Agrobacterium strain SSB64 has an ANI value significantly lower than 96% with both SSB39 and SSB74, and is therefore considered to be a different species from these two strains.

[0019] The Agrobacterium bacterium described above may have an ANI value of 97% or more, 98% or more, or 99% or more of the base sequence of its genomic DNA relative to the base sequence of the genomic DNA shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. Preferably, the Agrobacterium bacterium described above has the genomic DNA shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, i.e., is the Agrobacterium bacterium SSB39 strain, SSB64 strain, or SSB74 strain.

[0020] Another embodiment of the present invention is the Agrobacterium genus SSB16 strain (Accession No. NITE AP-04005), Agrobacterium genus SSB19 strain (Accession No. NITE AP-04042), Rhizobium genus SSB22 strain (Accession No. NITE AP-04007), Rhizobium genus SSB50 strain (Accession No. NITE AP-04043), or Agrobacterium genus SSB62 strain (Accession No. NITE AP-04009). These genus names were identified by 16s rRNA sequence analysis.

[0021] The Agrobacterium SSB16, Rhizobium SSB22, and Agrobacterium SSB62 strains were deposited on November 6, 2023, and the Agrobacterium SSB19 and Rhizobium SSB50 strains were deposited on December 12, 2023, at the NPMD National Institute of Technology and Evaluation (NPMD) Patent Microorganisms Deposit Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818).

[0022] The eight Agrobacterium or Rhizobium bacterial strains described above were isolated from soybean plants of the Peking variety, which are tolerant to waterlogging. The Agrobacterium or Rhizobium bacteria of the present invention have the effect of reducing waterlogging stress in soybeans (Glycine max (L.) Merrill). In this specification, reducing waterlogging stress in soybeans specifically refers to, for example, suppressing a decrease in soybean emergence rate and / or poor growth after germination due to waterlogging.

[0023] As will be described in detail in the Examples below, it has been found that when the Agrobacterium or Rhizobium bacterium according to the present invention is applied during cultivation to soybean varieties with low tolerance to flooding damage, it suppresses a decrease in the germination rate and poor growth caused by flooding stress in soybeans. The Agrobacterium or Rhizobium bacterium according to the present invention can reduce flooding stress even in varieties that do not genetically have flooding stress tolerance, without the need for genetic improvement of soybeans.

[0024] It is known that soybean rhizobia (bacteria of the genus Bradyrhizobium) contribute to the growth of soybeans by forming nodules from the middle stage of growth onwards when the soybean roots have grown to a certain extent. On the other hand, it has been surprisingly confirmed that the Agrobacterium or Rhizobium bacteria of the present invention are also effective at the time of germination and in the early stage of growth immediately after germination.

[0025] In regions of Japan that experience the rainy season, the soybean sowing time and early growth period tend to overlap with the rainy season, which means that flooding or inundation of soybean fields can easily cause moisture damage, such as a decrease in soybean emergence rate and poor growth. The Agrobacterium or Rhizobium bacterium of the present invention can suppress the decrease in soybean emergence rate and poor growth caused by flooding or inundation of fields, and therefore it is expected that using the Agrobacterium or Rhizobium bacterium of the present invention in soybean cultivation will suppress moisture damage in soybeans and improve soybean yield. The Agrobacterium or Rhizobium bacterium of the present invention may also be applied to soybeans grown under conditions without flooding stress.

[0026] The soybean variety to which the Agrobacterium or Rhizobium bacterium of the present invention is applied is not particularly limited, but is particularly effective in varieties that are considered to have no or low tolerance for moisture damage. The soybean variety may be a Japanese domestic variety or a variety from another country. The soybean variety to which the Agrobacterium or Rhizobium bacterium of the present invention is applied may be, for example, a variety other than the Pekin variety, such as Aomarukun, Akitamidori, Akimaro, Akimiyabi, Ayakogane, Ayamidori, Iwaikuro, Elstar, Enrei, Enreinosora, Osuzu, Osodenomai, Ootsuru, Okushirome, Otofuke Osode, Kitamusume, Kinusayaka, and Kiyo. Midori, Ginrei, Kurokojiro, Kuro Sayaka, Kurodamaru, Kuromaru-kun (Tohoku 161), Kogane Sayaka, Kosuzu, Kotoyutaka, Kotoyutaka A1 (Kanto 122), Sachiyutaka, Sachiyutaka A1, Village Smile, Shuryu, Shurei, Shintanba Kuro, Suzuotome, Suzukaori, Suzukaren, Suzukogane, Suzu Sayaka, Suzunooto, Suzuhonoka, Suzuho Rare, Suzumaru, Suzumaru R (Nakaiku No. 69), Suzuyutaka, Suzuroman, Tachinagaha, Tachiyutaka, Tatsumaro, Tamaurara, Tamadaikoku, Tamafukura, Tamahomare, Tanbaguro (Military Black No. 3), Tanrei, Chikushi B5, Tsubuhomare, Tsurumusume, Tomoyutaka, Toyokomachi, Toyoharuka, Toyomadoka (Jiku No. 258), Toyomizuki, Toyomusume, Nakasennari, Natto small grains, Nanahomare, Hatamusume, Hatayutaka, Hatsusayaka, Hatsunaga, Hana Daikoku, Hayahikari, Fukuakane (Kyushu No. 171), Fukuibuki, Fukuhayate, Fukuminori, Fukuyutaka, Fukuyutaka A1, Hoshi no Megumi (Higashiyama No. 228), Miyagishirome, Murayutaka, Yukishizuka, Yukipirika, Yukihomare, Yukihomare R, Yumenozuru, Yumeminori, Ryuho, etc.

[0027] The Agrobacterium or Rhizobium bacteria according to the present invention can be cultured by conventional methods. LB medium, for example, can be used as the culture medium. The culture temperature may be, for example, 10 to 30°C, preferably 20 to 30°C. The culture time may be, for example, 16 to 48 hours.

[0028] (Treatment agent) One embodiment of the present invention is a treating agent for soybean cultivation comprising at least one bacterium of the genus Agrobacterium or Rhizobium according to the present invention. Because the treating agent for soybean cultivation according to this embodiment comprises the bacterium of the genus Agrobacterium or Rhizobium according to the present invention, when used in soybean cultivation, it can reduce waterlogging stress in soybeans.

[0029] The soybean cultivation treatment agent can be applied to the cultivation area, such as soil, before, during, or during soybean growth. It may also be applied directly to soybean seeds before or during sowing, or to soybean plants during development. When applied to the cultivation area or soybean plants during soybean growth, the application time may be, for example, early in the soybean growth stage, within 10, 7, 5, or 2 days after sowing. Furthermore, by applying the soybean cultivation treatment agent to soybean seeds or soybean plants, it is possible to harbor the Agrobacterium or Rhizobium bacterium of the present invention within the soybeans, and the Agrobacterium or Rhizobium bacterium of the present invention can then be harbored within the next-generation soybean seeds obtained by subsequent growth. The soybean seeds harboring the Agrobacterium or Rhizobium bacterium thus obtained can be further cultivated to reduce flooding stress during the growth of the soybean seeds.

[0030] When the soybean cultivation treatment agent is applied to a cultivation area or to growing soybean plants, the soybean cultivation treatment agent may be applied as is, for example, by spraying or mixing with soil, or may be applied after dilution, or may be added to water used during watering.

[0031] Methods for applying the soybean cultivation treating agent to soybean seeds before sowing include, for example, immersing the soybean seeds in a liquid in which the treating agent has been previously suspended or in the treating agent in suspension form, spraying the soybean seeds with the treating agent in powder or liquid form, etc. When soybean seeds are immersed in a suspension, the immersion time may be, for example, about 30 seconds to 1 minute.

[0032] The soybean cultivation treating agent according to this embodiment may consist solely of at least one fungal cell of the Agrobacterium or Rhizobium bacterium according to the present invention, and may be formulated in the form of, for example, a dust, granule, wettable powder, liquid, emulsion, oil, suspension, paste, capsule, or aerosol. In addition to the Agrobacterium or Rhizobium bacterium according to the present invention, the soybean cultivation treating agent may contain, for example, a carrier, excipient, surfactant, binder, dispersant, preservative, antioxidant, etc., and may also contain a culture medium in which the fungal cells have been cultured. Furthermore, the soybean cultivation treating agent may contain a solid medium such as vermiculite, perlite, potting soil, or peat moss, or a liquid medium such as liquid fertilizer.

[0033] The amount of the Agrobacterium or Rhizobium bacterium according to the present invention contained in the treating agent for soybean cultivation is, for example, 1 × 10 0 cfu / ml ~ 1 × 10 12 cfu / ml, 1 × 10 1 cfu / ml ~ 1 × 10 11 cfu / ml, or 1 x 10 2 cfu / ml ~ 1 × 10 10 The amount of the Agrobacterium or Rhizobium bacterium according to the present invention contained in the soybean cultivation treatment agent 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, 99% by mass or more, or 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, relative to the total amount of the soybean cultivation treatment agent.

[0034] (soil) One embodiment of the present invention is a method for producing soil for soybean cultivation, which comprises adding at least one bacterium of the genus Agrobacterium or Rhizobium according to the present invention to soil. Because the soil for soybean cultivation obtained by the production method according to this embodiment contains the bacterium of the genus Agrobacterium or Rhizobium according to the present invention, using this soil in a cultivation area for soybeans can reduce waterlogging stress on soybeans.

[0035] In the soil for soybean cultivation according to this embodiment, it is preferable that the Agrobacterium or Rhizobium bacterium according to the present invention is uniformly mixed into the soil. The Agrobacterium or Rhizobium bacterium may be added as the above-mentioned soybean cultivation treatment agent. The soil used for soybean cultivation may be one that is commonly used for soybean cultivation. The amount of the Agrobacterium or Rhizobium bacterial strain contained in the soil for soybean cultivation is, for example, 1 × 10 0 cfu / ml ~ 1 × 10 12 cfu / ml, 1 × 10 1 cfu / ml ~ 1 × 10 11 cfu / ml, or 1 x 10 2 cfu / ml ~ 1 × 10 10 It may be cfu / ml.

[0036] The soil for soybean cultivation according to this embodiment may be used as is for cultivating soybeans, or may be mixed with other soil and used for cultivating soybeans.

[0037] (soybean seeds) One embodiment of the present invention is a method for producing soybean seeds for cultivation, which comprises attaching and / or encapsulating at least one Agrobacterium or Rhizobium bacterium of the present invention to soybean seeds. By attaching and / or encapsulating the Agrobacterium or Rhizobium bacterium of the present invention to soybean seeds, the obtained soybean seeds for cultivation can have the Agrobacterium or Rhizobium bacterium of the present invention on their surface, inside, or both. Therefore, by sowing the soybeans as they are, soybeans can be simply cultivated in a state in which the Agrobacterium or Rhizobium bacterium of the present invention coexists, and waterlogging stress during soybean seed germination and growth can be reduced.

[0038] Adhering and / or encapsulating the Agrobacterium or Rhizobium bacterium of the present invention to soybean seeds can be achieved, for example, by contacting the Agrobacterium or Rhizobium bacterium of the present invention with soybean seeds. Contacting soybean seeds with the Agrobacterium or Rhizobium bacterium of the present invention can be achieved, for example, by preparing a suspension of the Agrobacterium or Rhizobium bacterium of the present invention in advance and immersing the soybean seeds in the suspension, or by spraying the soybean seeds with a powder containing the suspension or the Agrobacterium or Rhizobium bacterium of the present invention. Furthermore, applying Agrobacterium or Rhizobium bacterium to soybean plants allows the bacterium to grow endophytically in the next generation of soybean seeds obtained by cultivating the plants. Therefore, applying the Agrobacterium or Rhizobium bacterium of the present invention to soybean plants allows the Agrobacterium or Rhizobium bacterium of the present invention to be encapsulated in soybean seeds. The Agrobacterium or Rhizobium bacterium according to the present invention may be attached to and / or encapsulated in soybean seeds as the above-mentioned agent for treating soybean cultivation or as one of its components.

[0039] The amount of the Agrobacterium or Rhizobium bacterium according to the present invention present on the surface and inside of soybean seeds for cultivation is, for example, 1 × 10 per soybean seed. 0 cfu ~ 1 × 1014 cfu, 1 × 10 1 cfu ~ 1 × 10 12 cfu, 1 × 10 2 cfu ~ 1 × 10 10 The soybean variety of the soybean seeds for cultivation is not particularly limited, and may be, for example, a variety other than the Pekin variety, such as any of the varieties described above.

[0040] (Cultivation method) One embodiment of the present invention is a method for cultivating soybeans, comprising applying at least one Agrobacterium or Rhizobium bacterium according to the present invention to cultivation land, soybean seeds, or soybean plants. The cultivation method according to this embodiment can reduce waterlogging stress in the soybeans being cultivated. Therefore, the cultivation method according to this embodiment can also be referred to as a method for reducing waterlogging stress in soybeans or a method for imparting waterlogging stress tolerance to soybeans.

[0041] The soybean cultivation area may be, for example, a field, a hydroponic cultivation area, a seedling raising tray, a seedling raising box, a seedling raising mat, or the like.

[0042] In the soybean cultivation method, the target of application, application timing, etc. of the Agrobacterium or Rhizobium bacteria of the present invention can be the same as those in the case of the above-mentioned treating agent for soybean cultivation. The Agrobacterium or Rhizobium bacteria can be applied to a cultivation area, soybean seeds, or soybean plants by applying the above-mentioned treating agent for soybean cultivation to the target. The Agrobacterium or Rhizobium bacteria can be applied to a cultivation area by using the above-mentioned soil for soybean cultivation in the cultivation area. [Example]

[0043] 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.

[0044] [1. Flooding stress induction test] The effect of soybean seed soaking time on growth was investigated using the following method. Enrei soybean seeds were soaked for the specified period in a petri dish filled with water. The soaked soybean seeds were sown 1 cm deep in pots filled with culture soil. The culture soil used was a 1:1 mixture of Super Mix (Sakata Seed Co., Ltd.) and vermiculite. The soybean growth status was investigated by measuring the soybean emergence rate, the above-ground part length 7 days after sowing, and the underground part length.

[0045] The soybean growth was examined by changing the soybean soaking time before sowing from 0.5 to 24 hours, and it was confirmed that soybean soaking for 4 hours reduced growth to the same extent as soybean soaking for 24 hours. Therefore, in subsequent tests, the soybean soaking time was set at 4 hours to subject soybeans to soaking stress before sowing.

[0046] 2. Isolation of Microorganisms Peking soybean seeds, which are tolerant to waterlogging, were soaked in distilled sterilized water for 18 hours or not soaked, and then sown in pots containing culture soil. Five days after sowing, the plants were sampled, separated into aboveground and underground parts, and disrupted in distilled sterilized water using a micro-smash bead cell disrupter (Tomy Seiko). The resulting suspension was serially diluted and plated on LB medium. Ninety-six strains were randomly selected from the resulting colonies, cultured in LB medium, and stored at -80°C. DNA was extracted, and the 16s rRNA sequence was decoded using a Sanger sequencer. The approximate species was identified using a BLAST search.

[0047] [3. Water Immersion Stress Test (1)] The ability of microorganisms to alleviate flooding stress was investigated. Among the endophytic microorganisms isolated from Pekin rice as described above, Agrobacterium cavallae 1-37, Agrobacterium sp. SSB39, Agrobacterium sp. SSB64, Agrobacterium sp. SSB74, and Agrobacterium larimorei 1-86 were selected. Stenotrophomonas maltophila RSB2, an endophytic microorganism isolated from rice, was also used in the study. While the 16s rRNA sequence analysis of Agrobacterium sp. SSB39, Agrobacterium sp. SSB64, and Agrobacterium sp. SSB74 all identified them as belonging to the genus Rhizobium, whole-genome analysis (described below) identified them as new species belonging to the genus Agrobacterium.

[0048] Bacterial suspensions were prepared by suspending each type of bacteria in distilled, sterilized water at an OD of 0.1 (wavelength: 600 nm). Soybean seeds of the Enrei variety, a variety that is not tolerant of waterlogging, were soaked in each suspension for 4 hours. The emergence rate and the lengths of the aboveground and underground parts 7 days after sowing were examined using the same method as in the flooding stress induction test described above. The emergence rate was expressed as the number of germinated seeds out of nine seeds sown in the same test plot. For comparison, soybean seeds soaked in distilled, sterilized water without bacterial suspension for 5 minutes or 4 hours, as well as unsoaked soybean seeds, were similarly examined for growth. The results of the emergence rate are shown in Table 1. The aboveground length and underground length 7 days after sowing are shown in Figure 1 and Figure 2, respectively.

[0049] [Table 1]

[0050] Soybean seeds treated for 4 hours with a flooding solution containing no bacteria showed a significantly reduced emergence rate and impaired shoot and root growth compared with untreated control plots, confirming flooding stress. Among the plots containing bacteria in the flooding solution, those containing Agrobacterium cavallae 1-37, Agrobacterium larrymorei 1-86, or Stenotrophomonas maltophila RSB2 showed no significant recovery in emergence rate, shoot length, or root length compared with the bacteria-free 4-hour flooding plots. On the other hand, soybean seeds soaked for 4 hours in a flooding solution containing Agrobacterium sp. SSB39, SSB64, or SSB74 showed a significant recovery in emergence rate and improved shoot and root length compared with the bacteria-free 4-hour flooding plots. It has been confirmed that the Agrobacterium strains SSB39, SSB64, and SSB74 have the ability to alleviate the reduced seedling emergence rate and growth inhibition caused by flooding stress in soybeans.

[0051] [4. Whole genome sequence analysis] The whole genome sequences of Agrobacterium strains SSB39, SSB64, and SSB74 were decoded using a nanopore sequencer (Oxford Nanopore, PromethION). The genomes were assembled by de novo assembly using the tool dragonflye1.0.13 (https: / / github.com / rpetit3 / dragonflye).

[0052] The genome of each strain, consisting of three chromosomes and a plasmid, was obtained. Details are shown in Table 2. [Table 2]

[0053] Using the decoded genome information, annotation was performed using the DDBJ DFAST pipeline (https: / / dfast.ddbj.nig.ac.jp / ). Taxonomy checks were performed using FastANI (Jain, et al. 2018), which is included in the pipeline. If the ANI value exceeds 96%, it is determined to be the same species. As a result, none of the three strains had an ANI value exceeding 96% with any known species.

[0054] The ANI values ​​(%) of the genomic DNA of each strain were also calculated. The results are shown in Table 3. The SSB39 and SSB74 strains were identified as belonging to the same species because their ANI values ​​exceeded 96%. Furthermore, the SSB64 strain was identified as belonging to a different species from the SSB39 and SSB74 strains because its ANI value was lower than these two strains.

[0055] [Table 3]

[0056] We also analyzed the strains using Type (Strain) Genome Server (https: / / tygs.dsmz.de), which covers a larger number of known bacterial species. As a result, similar to the FastANI analysis described above, all three strains were determined to be new species belonging to the genus Agrobacterium.

[0057] [5. Water Immersion Stress Test (2)] Endophytic microorganisms isolated from Peking soybean (S. cerevisiae) strains SSB16, SSB19, SSB20, SSB22, SSB50, SSB62, and 1-96 were used to test the effects of strains SSB16, SSB19, SSB20, SSB22, SSB50, SSB62, and 1-96 on Enrei soybean seeds to mitigate flooding stress. Bacterial suspensions were prepared by suspending each bacterial strain in distilled, sterilized water at an OD of 0.1 (wavelength: 600 nm). Enrei soybean seeds were submerged in each suspension for 5 hours, and the emergence rate and shoot and root lengths 7 days after sowing were determined using the same method as in the flooding stress induction test described above. Nine seeds were sown in each pot in triplicate for each test plot (27 seeds in total per test plot). For comparison, soybean seeds were submerged in distilled water for 5 hours without the bacteria suspension, and their growth was examined. The emergence rate for each pot is shown in Table 4, and the emergence rate (%) is shown in Figure 3. The above-ground length of the soybean plant body 7 days after sowing is shown in Figure 4, and the underground length is shown in Figure 5.

[0058] [Table 4]

[0059] 16srRNA sequence analysis revealed that the SSB16 and SSB20 strains were identical. Furthermore, 16srRNA sequence analysis identified the SSB16, SSB19, and SSB62 strains as Agrobacterium, the SSB22 and SSB50 strains as Rhizobium, and the 1-96 strain as Pseudomonas.

[0060] Among the test plots in which bacteria were added to the soaking treatment solution, the test plots using SSB16 (SSB20), SSB19, SSB22, SSB50, and SSB62 strains showed a clear recovery in emergence rate compared to the test plots treated with a 4-hour soaking treatment without bacteria, and growth was also recovered in both above-ground and below-ground length, confirming that the treatment has the effect of alleviating the decline in emergence rate and growth inhibition caused by soybean soaking stress. [Accession number]

[0061] (1) Name of depository institution: NPMD National Institute of Technology and Evaluation, Patent Microorganism Deposit Center (2) Contact: 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818 (3) Receipt number or accession number: NITE P-03789, NITE P-03790, NITE P-03791, NITE AP-04005, NITE AP-04042, NITE AP-04007, NITE AP-04043, NITE AP-04009 (4) Identification markings: SSB39, SSB64, SSB74, SSB16, SSB19, SSB22, SSB50, SSB62 (5) Date of receipt or acceptance: December 7, 2022 (NITE P-03789, 03790, 03791), November 6, 2023 (NITE AP-04005, 04007, 04009), December 12, 2023 (NITE AP-04042, NITE AP-04043)

Claims

1. Agrobacterium sp. strain SSB39 (accession number NITE P-03789), Agrobacterium sp. strain SSB64 (accession number NITE P-03790), Agrobacterium sp. strain SSB74 (accession number NITE P-03791), Agrobacterium sp. strain SSB16 (accession number NITEP-04005), Agrobacterium sp. strain SSB19 (accession number NITEP-04042), Rhizobium sp. strain SSB22 (accession number NITEP-04007), Rhizobium sp. strain SSB50 (accession number NITEP-04043), or Agrobacterium sp. strain SSB62 (accession number NITEP-04009).

2. A soybean cultivation treatment agent comprising at least one strain according to claim 1.

3. 10. A method for producing soil for growing soybeans, comprising adding to the soil at least one strain of the fungus according to claim 1.

4. 10. A method for producing soybean seeds for cultivation, comprising attaching and / or encapsulating at least one strain of the strain according to claim 1 to soybean seeds.

5. 10. A method for cultivating soybeans, comprising applying at least one strain of claim 1 to the cultivation field, soybean seeds or soybean plants.

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