Rhizobium and method for infecting leguminous plant with rhizobium

Abstract

Provided is a rhizobium in which the function of at least one effector gene for inducing symbiotic incompatibility with a leguminous plant is reduced or lacking.

Description

Rhizobium and method for infecting legumes with rhizobium

[0001] The present invention relates to rhizobia and a method for infecting legume plants with rhizobia.

[0002] Rhizobium infects legumes such as soybeans and forms nodules. Inside the nodules, the infected rhizobium fixes atmospheric nitrogen, which is then supplied to the host plant as nitrogen nutrition, promoting its growth. Therefore, efforts have been made to improve the production of host plants by spraying beneficial rhizobium, which have a high growth-promoting effect on host plants, into fields as microbial materials. Non-Patent Document 1 discloses the growth of soybeans under different cultivation temperature environments, the effect of inoculation with beneficial soybean rhizobium, and the impact on the community structure of infected soybean rhizobium.

[0003] S. Shiro, et al. "Effects of cultivation temperature on soybean growth and the community structure of infected soybean rhizobia." Japanese Journal of Soil Science and Plant Nutrition 92.3 (2021): 255-262.

[0004] The present inventors have found that when beneficial rhizobia are introduced into a field as a microbial material, they lose out in infection competition with indigenous rhizobia present in the field during the process of infecting legume plants, and are therefore unable to form sufficient nodules. In view of the above circumstances, an object of the present invention is to provide a method for improving the infectivity of a target rhizobia to legume plants, and to provide rhizobia to be used in said method.

[0005] As a result of extensive research, the inventors discovered that the infectivity of a target rhizobia to a legume plant expressing two or more symbiotic incompatibility genes can be improved by inoculating the legume plant with a target rhizobia that lacks or has reduced function in at least one effector gene that induces symbiotic incompatibility with the legume plant, and thus completed the present invention.

[0006] The present disclosure relates to, for example, the following [1] to

[14] . [1] A rhizobia in which the function of at least one effector gene that induces symbiotic incompatibility with legumes is deleted or reduced. [2] The rhizobia according to [1], which is a bacterium of the genus Bradyrhizobium. [3] The rhizobia according to [1] or [2], which is Bradyrhizobium ottawaense. [4] The rhizobia according to any of [1] to [3], in which the deletion of the function of the gene is due to a transposon insertion in the NopP gene or a mutation in the amino acid sequence of NopP. [5] The rhizobia according to [4], wherein the mutation in the amino acid sequence of NopP is due to a substitution of the amino acid residue corresponding to position 60 in the amino acid sequence of SEQ ID NO: 2 with leucine and the amino acid residue corresponding to position 271 with phenylalanine. [6] The rhizobia according to any of [1] to [5], wherein NopP is a protein in which the amino acid residue corresponding to position 60 in SEQ ID NO: 2 is substituted with leucine and the amino acid residue corresponding to position 271 with phenylalanine in the FY2-m1 strain (accession number NITE BP-04046), GMA461-m4 strain (accession number NITE BP-04047), OSA024 strain (accession number NITE BP-04048), or Bradyrhizobium ottawaense SG09 strain (accession number NITE BP-03361). [7] A method for infecting a legume plant with rhizobia, comprising inoculating a legume plant expressing two or more symbiotic incompatibility genes with rhizobia in which the function of at least one effector gene that induces symbiotic incompatibility with the legume plant is deleted or reduced. [8] The method according to [7], wherein the rhizobia is a bacterium of the genus Bradyrhizobium. [9] The method according to [7] or [8], wherein the rhizobia is Bradyrhizobium ottawaense.

[10] The method according to any of [7] to [9], wherein the deletion of the gene function is due to transposon insertion in the NopP gene or due to a mutation in the amino acid sequence of NopP.

[11] The method according to

[10] , wherein the mutation in the amino acid sequence of NopP is due to a substitution of the amino acid residue corresponding to position 60 in the amino acid sequence of SEQ ID NO: 2 with leucine and the amino acid residue corresponding to position 271 with phenylalanine.

[12] The method according to any of [7] to

[11] , wherein the rhizobia is a strain of FY2-m1 (Accession No. NITE BP-04046), GMA461-m4 (Accession No. NITE BP-04047), OSA024 (Accession No. NITE BP-04048), or Bradyrhizobium ottawaense SG09 (Accession No. NITE BP-03361), in which NopP is a protein in which the amino acid residue corresponding to position 60 in SEQ ID NO: 2 is substituted with leucine and the amino acid residue corresponding to position 271 with phenylalanine.

[13] A method for producing the rhizobia according to any one of [1] to [6].

[14] A composition for infecting legumes, comprising one or more species of rhizobia according to any one of [1] to [6].

[0007] According to the present invention, it is possible to provide a method for improving the infectivity of a target rhizobia to legume plants, and a rhizobia to be used in the method.

[0008] (A) A diagram showing the results of electrophoresis of the Rj2 and GmNNL1 gene markers amplified using DNA extracted from soybean as a template. (B) A diagram showing the results of electrophoresis of the amplified fragment of the above gene region. (C) A diagram showing the positional relationship between the above gene region and the forward and reverse primers. (A) A diagram showing the position of transposon insertion in the nopP_122 gene of the Bradyrhizobium ottawaense FY2-m1 and GMA461-m4 strains, and the Bradyrhizobium diazoefficiens W3-1a and W9-1a strains. (B) A diagram showing the results of electrophoresis of the amplified fragment of the above gene region. (A) A graph showing the number of mature nodules and a photograph of the nodulated roots when Rj2 / GmNNL1 gene-integrated soybeans were inoculated with Bradyrhizobium ottawaense GMA461 or GMA461-m4 strain. (B) A graph showing the number of mature nodules and a photograph of the nodulated roots when Rj2 / GmNNL1 gene-integrated soybeans were inoculated with Bradyrhizobium ottawaense FY2 or FY2-m1 strain. (C) A graph showing the number of mature nodules and a photograph of the nodulated roots when Rj2 / GmNNL1 gene-integrated soybeans were inoculated with Bradyrhizobium diazoefficiens USDA122, W3-1a, or W9-1a strain. Exemplary band patterns of restriction enzyme fragments of each nopP gene function-losing strain and each nopP gene-carrying strain. 1 is a graph showing the total number of mature nodules formed on Akutashiraji soybean and the number of mature nodules occupied by each nopP gene function-loss strain. (A) SG09 strain or SG09nopP ＬＦ (B) The band patterns of restriction enzyme fragments of the amplified fragments of the gene regions of the strains. ＬＦ(A) A graph showing the nodule occupancy of each strain when Rj2 / GmNNL1 soybean was inoculated with the GMA461-m4 strain or each nopP gene-carrying strain in the field. (B) A graph showing the nodule occupancy of each strain when Rj2 / GmNNL1 soybean was inoculated with the GMA461-m4 strain or each nopP gene-carrying strain in the field. ２ 1 is a graph showing the total number of mature nodules formed on rj2 / Gmnnl1 soybean and the number of mature nodules occupied by each nopP gene function-loss strain. 2 is a graph showing the total number of mature nodules formed on Rj2 / GmNNL1 soybean and the number of mature nodules occupied by each nopP gene function-loss strain. (A) N2O flux when Rj2 / GmNNL1 soybean or rj2 / Gmnnl1 soybean was administered a microbial material containing the nopP gene function-loss strain GMA461-m4 and the nopP gene-carrying strains USDA6, USDA110, and USDA122 and subjected to nodule aging treatment. ２ (B) The amount of N released when Rj2 / GmNNL1 soybean or rj2 / Gmnnl1 soybean was treated with a microbial material containing the nopP gene function-deficient strain FY2-m1 and the nopP gene-carrying strains USDA6, USDA110, and USDA122, and then subjected to nodule senescence treatment. ２ (C) The amount of O released when Rj2 / GmNNL1 soybean or rj2 / Gmnnl1 soybean was treated with a microbial material containing the nopP gene function-deficient strain OSA024 and the nopP gene-carrying strains USDA6, USDA110, and USDA122, and then subjected to nodule senescence treatment. ２(A) A graph showing the nodule occupancy rate of each strain when Rj2 / GmNNL1 soybeans were sown in field soil collected on the premises of the National Agriculture and Food Research Organization (hereinafter also referred to as "NARO field soil") and a microbial material containing the GMA461-m4, FY2-m1, or OSA024 strain was administered. (B) A graph showing the NO release rate measured at various days after Rj2 / GmNNL1 soybeans were sown in NARO field soil and a microbial material containing the GMA461-m4, FY2-m1, or OSA024 strain was administered, followed by excision of the above-ground parts of the soybeans. ２ (C) Rj2 / GmNNL1 soybeans were seeded in soil from a farm operated by the National Agriculture and Food Research Organization, and a microbial material containing the GMA461-m4, FY2-m1, or OSA024 strain was administered. The above-ground parts of the soybeans were then excised, and the amount of O released was measured at various times. ２ 1 is a graph showing the total amount of O released.

[0009] Hereinafter, embodiments of the present disclosure will be described in detail.

[0010] [Method for infecting legume plants with rhizobia] The method for infecting legume plants with rhizobia according to this embodiment comprises inoculating a legume plant expressing two or more symbiotic incompatibility genes with rhizobia (hereinafter also referred to as "target rhizobia") in which the function of at least one effector gene that induces symbiotic incompatibility with the legume is deleted or reduced.

[0011] As used herein, "symbiotic incompatibility" means that infection of a legume plant with a specific genetic type by a rhizobia of a specific genetic type is suppressed or eliminated. Symbiotic incompatibility is triggered, for example, when a resistance protein gene (hereinafter also referred to as "R gene") expressed by the legume plant recognizes an effector injected into the legume plant when the rhizobia infect the legume plant, causing a resistance reaction.

[0012] In this specification, the term "nodule" refers to a "mature nodule" that has been infected with rhizobia, or a "nodule-like structure" that has not been infected with rhizobia. Whether or not a nodule is a mature nodule may be determined if the inside of the nodule is pink to red. Whether or not a nodule is a nodule-like structure may be determined if the inside of the nodule is white.

[0013] The above method can improve the infectivity of a target rhizobia to a legume. For example, after inoculating a target rhizobia into the legume and cultivating the legume, the infectivity of the target rhizobia can be determined to have been improved if the number of mature nodules formed on the roots is greater than the number of mature nodules formed by the same procedure except that an effector gene-carrying control is inoculated instead of the target rhizobia, for example, by at least two, three, four, five, ten, or twenty times. Examples of situations in which the infectivity of the target rhizobia can be determined to have been improved include situations in which the effector gene-carrying control forms mature nodules but in fewer numbers than the target rhizobia, and situations in which the effector gene-carrying control forms nodule-like structures but hardly or not at all forms mature nodules, and the number of mature nodules is smaller than that of the target rhizobia.

[0014] The "control with effector gene" refers to a rhizobia carrying an effector gene that induces symbiotic incompatibility with the legume. The "control with effector gene" may be a rhizobia that is identical to the target rhizobia except that it lacks or has reduced function of at least one effector gene that induces symbiotic incompatibility with the legume, or it may be the wild-type rhizobia if the target rhizobia has been produced (created) by mutagenesis or genetic recombination of a wild-type rhizobia.

[0015] Furthermore, whether or not the infectivity of a target rhizobia to a legume plant has been improved can be determined based on whether or not the ratio of mature nodules occupied by the target rhizobia to all mature nodules of the legume plant (hereinafter also referred to as "nodule occupancy") can be increased when the target rhizobia and indigenous rhizobia compete for infection of the legume plant. The nodule occupancy can be determined, for example, by inoculating the target rhizobia and rhizobia expressing an effector gene that induces symbiotic incompatibility with the legume plant (hereinafter also referred to as "indigenous model bacteria") at the same bacterial counts into a legume plant expressing two or more symbiotic incompatibility genes, cultivating the legume plant, and then calculating the ratio of the number of mature nodules occupied by the target rhizobia to all mature nodules. More specifically, the nodule occupancy can be determined using the method described in Example 3.

[0016] Specifically, the indigenous model bacteria may be one, two, three, four or more types of rhizobia. These rhizobia are not particularly limited and known bacteria can be used. For example, when the legume expresses the GmNNL1 gene and the Rj2 gene, Bradyrhizobium japonicum USDA6 strain (hereinafter also referred to as the "USDA6 strain"; MAFF number: 211646), Bradyrhizobium diazoefficiens USDA110 strain (hereinafter also referred to as the "USDA110 strain"; JCM number: 10833), and Bradyrhizobium diazoefficiens USDA122 strain (hereinafter also referred to as the "USDA122 strain"; BCRC number: 13533) may be used. Furthermore, the "plurality of mature nodules" may be 25 or more, 50 or more, or 100 or more mature nodules.

[0017] The presence or absence of the target rhizobia and indigenous model bacteria in mature nodules can be confirmed, for example, by extracting DNA from mature nodules using standard methods, amplifying the effector gene region, treating it with restriction enzymes, subjecting it to electrophoresis, and confirming the band pattern based on restriction fragment length polymorphisms. The primer set and restriction enzymes used in the PCR method may be appropriately selected so that the band pattern of the target rhizobia can be distinguished from that of the indigenous model bacteria. When the effector gene is the nopP gene, the primer set used in the PCR method may include a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 14 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 15, and the restriction enzymes may be a combination of AluI and PstI. The presence or absence of the target rhizobia and indigenous model bacteria in mature nodules may also be confirmed by identifying the bacterial species based on the 16S rRNA gene or the like using DNA sequence analysis.

[0018] Whether or not the nodule occupancy rate of the target rhizobia has improved when the target rhizobia and the indigenous rhizobia are allowed to compete for infection of legumes can be determined, for example, by comparing the nodule occupancy rate determined for the target rhizobia with the nodule occupancy rate determined by the same procedure except that an effector gene-carrying control is used instead of the target rhizobia. For example, the nodule occupancy rate of the target rhizobia may be determined to have improved when the nodule occupancy rate determined for the target rhizobia is, for example, 1.1 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 10 times or more, or 20 times or more, compared to the nodule occupancy rate determined for the effector gene-carrying control.

[0019] Furthermore, when the nodule occupancy rate of a target rhizobia in soybeans carrying two or more symbiotic incompatibility genes is higher than the nodule occupancy rate of the target rhizobia determined using the same procedure except that soybeans not carrying the symbiotic incompatibility genes are used instead of soybeans carrying two or more symbiotic incompatibility genes, it can be determined that the nodule occupancy rate of the target rhizobia has improved when the target rhizobia and indigenous rhizobia compete for infection of legumes. For example, when the nodule occupancy rate of the target rhizobia in soybeans carrying two or more of the above-mentioned symbiotic incompatibility genes is 20 times or more, 25 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, 90 times or more, 100 times or more, 200 times or more, or 300 times or more compared to the nodule occupancy rate of the target rhizobia in soybeans not carrying the symbiotic incompatibility genes, it can be determined that the nodule occupancy rate of the target rhizobia is improved when the target rhizobia and indigenous rhizobia are allowed to compete for infection of a legume. Akutashirazu soybean variety or the like can be used as the soybean not carrying the above-mentioned symbiotic incompatibility genes.

[0020] When the target rhizobia and the indigenous rhizobia are allowed to compete for infection of a legume carrying two or more symbiotic incompatibility genes, if the nodule occupancy rate determined for the target rhizobia is 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more, it can be said that the target rhizobia is predominantly infecting the legume.

[0021] (Legume expressing two or more symbiotic incompatibility genes) In the present specification, the legume is not particularly limited as long as it is infected with rhizobia and forms root nodules, and examples thereof include plants of the genus Glycine soja (e.g., soybean, wild soybean, etc.), plants of the genus Medicago (e.g., alfalfa, etc.), plants of the genus Vitis (e.g., cowpea, adzuki bean, etc.), plants of the genus Phaseolus (e.g., Phaseolus vulgaris, etc.), plants of the genus Pisum sativum (e.g., winged bean, etc.), plants of the genus Vicia faba (faba bean, etc.), plants of the genus Jack bean (e.g., jack bean, etc.), plants of the genus Arachis hypogaea (e.g., peanut, etc.), plants of the genus Lentil (e.g., lentil, etc.), etc. In the method of infecting a legume with rhizobia according to this embodiment, the legume is preferably a plant of the genus Glycine soja (e.g., soybean, etc.), and more preferably soybean.

[0022] As used herein, soybean refers to Glycine max (L.) Merr. of the genus Glycine in 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.).

[0023] The symbiotic incompatibility gene is not particularly limited as long as it is a gene that causes symbiotic incompatibility with rhizobia that express an effector gene, and examples thereof include the NopP gene possessed by the Bradyrhizobium diazoefficiens USDA110 strain (hereinafter also referred to as the "NopP_110 gene"), the NopP gene possessed by the Bradyrhizobium japonicum USDA6 strain (hereinafter also referred to as the "NopP_6 gene"), and the Bradyrhizobium diazoefficiens USDA6 strain (hereinafter also referred to as the "NopP_6 gene"). The R gene may be a gene that induces symbiotic incompatibility with rhizobia that express at least one or more effector genes selected from the group consisting of the NopP gene possessed by the Sinorhizobium diazoefficiens USDA122 strain (hereinafter also referred to as the "NopP_122 gene"), the NopP gene possessed by the Sinorhizobium fredii USDA257 strain (hereinafter also referred to as the "NopP_257 gene"), and the Bel2-5 gene. When the legume plant is soybean, examples of the R gene include the GmNNL1 gene, the Rj2 gene, the Rfg1 gene, and the Rj4 gene.

[0024] Whether or not a gene (test gene) is a gene that induces symbiotic incompatibility with rhizobia that express an effector gene can be confirmed, without particular limitation, by inoculating a legume expressing the test gene and a legume in which the test gene has been knocked out with rhizobia that express an effector gene, and comparing the number of mature nodules formed after cultivation. For example, if the number of mature nodules formed after cultivation in the legume expressing the test gene is 0.7-fold or less, 0.5-fold or less, 0.3-fold or less, or 0.1-fold or less compared to the number in the legume in which the test gene has been knocked out, the test gene can be determined to be a gene that induces symbiotic incompatibility with rhizobia. Examples of situations in which it can be determined that the test gene is a gene that induces symbiotic incompatibility with the rhizobia include situations in which mature nodules are formed in legumes that express the test gene, but the number of nodules is smaller than in legumes in which the test gene has been knocked out, and situations in which nodule-like structures are formed in legumes that express the test gene, but few or no mature nodules are formed, and the number of mature nodules is smaller than in legumes in which the test gene has been knocked out.

[0025] The GmNNL1 gene is a gene consisting of the nucleotide sequence represented by SEQ ID NO: 16 and encodes a protein consisting of the amino acid sequence represented by SEQ ID NO: 17. The GmNNL1 gene induces symbiotic incompatibility with rhizobia that express the effector genes NopP_110 gene or NopP_6 gene (Zhang et al., "Glycine max NNL1 restricts symbiotic compatibility with widely distributed bradyrhizobia via root hair infection," Nature Plants (2021) 7, 73-86, WO 2022 / 135246).

[0026] The Rj2 gene is a gene consisting of the nucleotide sequence represented by SEQ ID NO: 18 and encodes a protein consisting of the amino acid sequence represented by SEQ ID NO: 19. The Rj2 gene induces symbiotic incompatibility with rhizobia that express the effector gene NopP_122 (Yang et al., "R gene-controlled host specificity in the legume-rhizobia symbiosis," Proceedings of the National Academy of Sciences (2010) 107, 18735-18740; Sugawara et al., "Variation in bradyrhizobial NopP effector determines symbiotic incompatibility with Rj2-soybeans via effector-triggered immunity," Nature Communications (2018) 9:3139).

[0027] The Rfg1 gene is a gene consisting of the nucleotide sequence represented by SEQ ID NO: 20, and is a different allele of the Rj2 gene. The Rfg1 gene encodes a protein consisting of the amino acid sequence represented by SEQ ID NO: 21. The Rfg1 gene induces symbiotic incompatibility with, for example, rhizobia that express the effector gene NopP_257 gene (Yang et al., "R gene-controlled host specificity in the legume-rhizobia symbiosis," Proceedings of the National Academy of Sciences (2010) 107, 18735-18740; Sugawara et al., Variation in bradyrhizobial NopP effector determines symbiotic incompatibility with Rj2-soybeans via effector-triggered immunity. Nature Communications (2018) 9:3139.).

[0028] The Rj4 gene is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 22 and encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 23. The Rj4 gene induces symbiotic incompatibility with rhizobia that express the effector gene Bel2-5 (Faruque, Omar M., et al. "Identification of Bradyrhizobium elkanii genes involved in incompatibility with soybean plants carrying the Rj4 allele", Applied and environmental microbiology 81.19 (2015): 6710-6717; Tang, Fang, et al. "Rj4, a gene controlling nodulation specificity in soybeans, encodes a thaumatin-like protein, but not the one previously reported", Plant Physiology 170.1 (2016): 26-32).

[0029] Other examples of symbiotic incompatibility genes include genes selected from the following (1) to (4): (1) (i) a gene consisting of a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 16, which induces symbiotic incompatibility with rhizobia that express an effector gene, (ii) a gene consisting of a base sequence in which 1 to 303 bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 16, which induces symbiotic incompatibility with rhizobia that express an effector gene, (iii) a gene consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, which encodes a protein that induces symbiotic incompatibility with rhizobia that express an effector gene, or (iv) a gene consisting of an amino acid sequence in which 1 to 101 amino acid residues are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 17, which encodes a protein that induces symbiotic incompatibility with rhizobia that express an effector gene. (2) (i) a gene consisting of a base sequence having 90% or more sequence identity with the base sequence represented by SEQ ID NO: 18, which induces symbiotic incompatibility with rhizobia that express an effector gene; (ii) a gene consisting of a base sequence in which 1 to 315 bases are deleted, substituted, or added in the base sequence represented by SEQ ID NO: 18, which induces symbiotic incompatibility with rhizobia that express an effector gene; (iii) a gene consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, which encodes a protein that induces symbiotic incompatibility with rhizobia that express an effector gene; or (iv) a gene consisting of an amino acid sequence in which 1 to 105 amino acid residues are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 19, which encodes a protein that induces symbiotic incompatibility with rhizobia that express an effector gene.(3) (i) a gene consisting of a base sequence having 90% or more sequence identity with the base sequence represented by SEQ ID NO: 20, which induces symbiotic incompatibility with rhizobia that express an effector gene; (ii) a gene consisting of a base sequence in which 1 to 315 bases are deleted, substituted, or added in the base sequence represented by SEQ ID NO: 20, which induces symbiotic incompatibility with rhizobia that express an effector gene; (iii) a gene consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 21, which encodes a protein that induces symbiotic incompatibility with rhizobia that express an effector gene; or (iv) a gene consisting of an amino acid sequence in which 1 to 105 amino acid residues are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 21, which encodes a protein that induces symbiotic incompatibility with rhizobia that express an effector gene. (4) (i) a gene consisting of a base sequence having 90% or more sequence identity with the base sequence represented by SEQ ID NO: 22, which induces symbiotic incompatibility with rhizobia; (ii) a gene consisting of a base sequence in which 1 to 89 bases are deleted, substituted, or added in the base sequence represented by SEQ ID NO: 22, which induces symbiotic incompatibility with rhizobia that express an effector gene; (iii) a gene consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 23, which encodes a protein that induces symbiotic incompatibility with rhizobia that express an effector gene; or (iv) a gene consisting of an amino acid sequence in which 1 to 29 amino acid residues are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 23, which encodes a protein that induces symbiotic incompatibility with rhizobia that express an effector gene.

[0030] The gene described in (1) may be a gene that causes symbiotic incompatibility with rhizobia that express the NopP_110 gene or the NopP_6 gene, the gene described in (2) may be a gene that causes symbiotic incompatibility with rhizobia that express the NopP_122 gene, the gene described in (3) may be a gene that causes symbiotic incompatibility with rhizobia that express the NopP_257 gene, and the gene described in (4) may be a gene that causes symbiotic incompatibility with rhizobia that express the Bel2-5 gene.

[0031] As used herein, "sequence identity" refers to the percentage (%) of identical bases or amino acid residues relative to the total overlapping DNA base sequences or total amino acid sequences in optimal alignment of two DNA base sequences or amino acid sequences using a mathematical algorithm known in the art. EMBOSS Needle (provided by EMBL-EBI, URL: https: / / www.ebi.ac.uk / tools / psa / emboss_needle / ) can be used, for example, to create the alignment and calculate the sequence identity.

[0032] In the genes (1) to (4), the sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0033] As used herein, base deletion means that some nucleotide bases are missing in a gene (DNA), resulting in a change (reduction) in the number of bases, and amino acid residue deletion means that some amino acid residues are deleted in a protein, resulting in a change (reduction) in the number of amino acids.

[0034] As used herein, base substitution means that a part of a nucleotide base in a gene (DNA) is replaced with another base. Amino acid residue substitution means that a part of an amino acid residue in a protein is replaced with another amino acid residue. Among single base pair substitutions, for example, silent mutations that do not result in a change in the amino acid and neutral mutations that result in a functionally identical amino acid are expected to have little effect on the transcriptional control function of the protein encoded by each gene.

[0035] In this specification, the term "addition (insertion) of a base" refers to the addition of a part of a nucleotide base in a gene (DNA) to change (increase) the number of bases, and the term "addition of an amino acid residue" refers to the addition of a part of an amino acid residue in a protein to change (increase) the number of amino acids.

[0036] In the genes (1) to (3), the number of bases to be deleted, substituted, or added may be, for example, 1 to 300, 1 to 250, 1 to 200, 1 to 150, 1 to 100, 1 to 50, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2.

[0037] In the gene (4), the number of bases deleted, substituted, or added may be, for example, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2.

[0038] In the genes of (1) to (3), the number of amino acid residues to be deleted, substituted, or added may be, for example, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2.

[0039] In the gene (4), the number of amino acid residues deleted, substituted, or added may be, for example, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2.

[0040] From the viewpoint of further improving the nodule occupancy rate of the target rhizobia, it is preferable to combine two or more symbiotic incompatibility genes so that symbiotic incompatibility can be exerted against two or more species of rhizobia expressing different effector genes. Since many rhizobia, including indigenous bacteria, express the NopP_110 gene, the NopP_6 gene, the NopP_122 gene, or the NopP_257 gene, from the viewpoint of further improving the nodule occupancy rate of the target rhizobia, a preferred combination of two or more symbiotic incompatibility genes is one that can exert symbiotic incompatibility against at least two or more species of rhizobia selected from the group consisting of rhizobia expressing the NopP_110 gene, rhizobia expressing the NopP_6 gene, and rhizobia expressing the NopP_122 gene and the NopP_257 gene.

[0041] The symbiotic incompatibility genes may be carried heterozygously or homozygously, but are preferably carried homozygously. It is more preferable that all of the two or more symbiotic incompatibility genes are carried homozygously.

[0042] A legume plant expressing a symbiotic incompatibility gene may be produced, for example, by incorporating the symbiotic incompatibility gene into the genomic DNA of the legume plant, or by modifying an existing gene to the symbiotic incompatibility gene using genome editing technology. Methods for incorporating the symbiotic incompatibility gene into the genomic DNA of a legume plant can be known methods, such as the Agrobacterium method and particle bombardment. Genome editing techniques can be known methods, such as transcription activator-like nuclease (TALEN) and CRISPR-Cas9. Whether or not the genomic DNA of a legume plant contains a symbiotic incompatibility gene may be confirmed by DNA sequence analysis.

[0043] Legumes expressing two or more symbiotic incompatibility genes can be produced, for example, by crossing legumes expressing different symbiotic incompatibility genes. Legumes homozygously carrying two or more symbiotic incompatibility genes can also be produced, for example, by first crossing a legume expressing a symbiotic incompatibility gene (first gene) with a legume expressing a symbiotic incompatibility gene (second gene) different from the symbiotic incompatibility gene, and then selecting F1 seeds heterozygously carrying the first gene and the second gene from the F2 seeds obtained from the F1 seeds. Preferably, the F2 seeds are crossed to obtain F3 seeds. The crossing of legumes may be carried out by cultivating them so that their flowering periods are the same, or by pollinating the emasculated buds of the maternal plant with pollen from the paternal plant.

[0044] Whether the symbiotic incompatibility gene is possessed in a heterozygous or homozygous state can be confirmed, for example, by extracting DNA from a legume by a conventional method, performing PCR using the DNA as a template with a primer set designed to distinguish the amplified fragment lengths of the dominant and recessive alleles of the symbiotic incompatibility gene, and subjecting the result to electrophoresis. For example, such a primer set may be a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 26 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 27 when the symbiotic incompatibility gene is the GmNNL1 gene, or a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 24 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 25 when the symbiotic incompatibility gene is the Rj2 gene.

[0045] Examples of legume varieties that express symbiotic incompatibility genes include soybean varieties that express the GmNNL1 gene, such as WC108 Uma (NARO Genebank Seed Control Number: JP27584) and WC165 Uma (NARO Genebank Seed Control Number: JP30138); soybeans that express the Rj2 gene include Bonminori (NARO Genebank Seed Control Number: JP28493, Ministry of Agriculture, Forestry and Fisheries Variety Registration Number: Norin 26); soybeans that express the Rfg1 gene include Williams82 (NARO Genebank Seed Control Number: JP31043); and soybeans that express the Rj4 gene include Fukuyutaka (NARO Genebank Seed Control Number: JP29668).

[0046] (Rhizobium having a defective or reduced function of at least one effector gene that induces symbiotic incompatibility with legumes) As used herein, examples of rhizobium include bacteria of the genus Bradyrhizobium, Allorhizobium, Azorhizobium, Ensifer, Rhizobium, Sinorhizobium, Mesorhizobium, Methylobacterium, Blastobacter, Devosia, Burkholderia, and Ralstonia. In the method of infecting legumes with rhizobium according to this embodiment, Bradyrhizobium is preferred as the rhizobium.

[0047] Rhizobium can form mature nodules by infecting legumes, and inside the nodules, the rhizobium can fix atmospheric nitrogen molecules using nitrogenase and the like. The fixed nitrogen is supplied to the legumes, promoting their growth. Therefore, rhizobium with high nitrogen fixation ability is useful from the perspective of increasing the yield of legumes. Furthermore, rhizobium carrying the nosZ gene can fix atmospheric dinitrogen monoxide (N ２ O) can be reduced to molecular nitrogen. ２ O is said to have a stronger greenhouse effect than carbon dioxide and to destroy the ozone layer. ２ Since it is said to excrete O, N ２ Rhizobium that can reduce O to molecular nitrogen, i.e., N２ Rhizobium with the ability to reduce oxygen is useful for environmental conservation, particularly in preventing global warming.

[0048] In this specification, examples of Bradyrhizobium bacteria include Bradyrhizobium ottawaense, Bradyrhizobium japonicum, Bradyrhizobium diazoefficiens, Bradyrhizobium elkanii, Bradyrhizobium canariense, Bradyrhizobium pachyrhizi, and Bradyrhizobium betae. In the method of infecting a legume plant with rhizobia according to this embodiment, the Bradyrhizobium bacterium is preferably Bradyrhizobium ottawaense or Bradyrhizobium diazoefficiens, and Bradyrhizobium ottawaense is more preferred from the viewpoint of further increasing the nodule occupancy rate. Bradyrhizobium ottawaense has a high N ２ It is also useful because it may have the ability to reduce nitrous oxide (Wasai-Hara, Sawa, et al., "Bradyrhizobium ottawaense efficiently reduces nitrous oxide through high nosZ gene expression," Scientific Reports 13.1 (2023): 18862).

[0049] In the method of infecting a legume with rhizobia according to this embodiment, the rhizobia (i.e., the target rhizobia) lack or have reduced function in at least one effector gene that induces symbiotic incompatibility with the legume. This allows the target rhizobia to efficiently infect the legume. When the target rhizobia expresses multiple effector genes that induce symbiotic incompatibility with the legume, it is preferable that the functions of all of the effector genes are lost or reduced.

[0050] As used herein, "effector" refers to a protein that is delivered from rhizobia to legumes via the rhizobia protein secretion apparatus. The protein secretion apparatus includes types III and IV. Effectors can also be referred to as type 3 secretion effectors or type 4 secretion effectors.

[0051] The "effector gene that induces symbiotic incompatibility with a legume" may be any effector gene that induces symbiotic incompatibility with a legume, and may be, for example, an effector gene that induces symbiotic incompatibility with a legume that expresses at least one R gene selected from the group consisting of the GmNNL1 gene, the Rj2 gene, the Rfg1 gene, and the Rj4 gene. Specific examples include the NopP gene and the Bel2-5 gene. The NopP gene has an amino acid polymorphism that is specific to each rhizobia strain. Examples of NopP genes include the NopP_110 gene, the NopP_6 gene, the NopP_122 gene, and the NopP_257 gene.

[0052] Whether or not an effector gene induces symbiotic incompatibility with a legume can be confirmed, without particular limitation, by inoculating the legume with rhizobia expressing the effector gene and rhizobia in which the effector gene has been knocked out, and comparing the number of mature nodules formed after cultivation. For example, if the number of mature nodules produced by rhizobia expressing the effector gene is 0.7-fold or less, 0.5-fold or less, 0.3-fold or less, or 0.1-fold or less compared to the number produced by rhizobia in which the effector gene has been knocked out, the effector gene can be determined to be an effector gene that induces symbiotic incompatibility with a legume. Examples of situations in which an effector gene can be determined to be an effector gene that induces symbiotic incompatibility with the legume include situations in which rhizobia expressing the effector gene form mature nodules, but the number of these nodules is smaller than the number of mature nodules in rhizobia in which the effector gene has been knocked out, and situations in which rhizobia expressing the effector gene form nodule-like structures, but few or no mature nodules are formed, and the number of these mature nodules is smaller than the number of mature nodules in rhizobia in which the effector gene has been knocked out.

[0053] The NopP_110 gene is a gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 and encodes a protein consisting of the amino acid sequence represented by SEQ ID NO: 2. The NopP_6 gene is a gene consisting of the nucleotide sequence represented by SEQ ID NO: 3 and encodes a protein consisting of the amino acid sequence represented by SEQ ID NO: 4. The NopP_122 gene is a gene consisting of the nucleotide sequence represented by SEQ ID NO: 5 and encodes a protein consisting of the amino acid sequence represented by SEQ ID NO: 6. The NopP_257 gene is a gene consisting of the nucleotide sequence represented by SEQ ID NO: 7 and encodes a protein consisting of the amino acid sequence represented by SEQ ID NO: 8. The Bel2-5 gene is a gene consisting of the nucleotide sequence represented by SEQ ID NO: 9 and encodes a protein consisting of the amino acid sequence represented by SEQ ID NO: 10.

[0054] Other examples of effector genes that induce symbiotic incompatibility with legumes include genes selected from the following (5) to (9): (5) (i) a gene that induces symbiotic incompatibility with legumes, the gene consisting of a nucleotide sequence having 60% or more sequence identity with the nucleotide sequence set forth in SEQ ID NO: 1, (ii) a gene that induces symbiotic incompatibility with legumes, the gene consisting of a nucleotide sequence in which 1 to 83 nucleotides are deleted, substituted, or added in the nucleotide sequence set forth in SEQ ID NO: 1, (iii) a gene that encodes a protein that induces symbiotic incompatibility with legumes, the gene consisting of an amino acid sequence having 60% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2, or (iv) a gene that encodes a protein that induces symbiotic incompatibility with legumes, the gene consisting of an amino acid sequence in which 1 to 27 amino acid residues are deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 2. (6) (i) a gene consisting of a base sequence having 60% or more sequence identity with the base sequence shown in SEQ ID NO: 3, which induces symbiotic incompatibility with legumes; (ii) a gene consisting of a base sequence in which 1 to 83 bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 3, which induces symbiotic incompatibility with legumes; (iii) a gene consisting of an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4, which encodes a protein that induces symbiotic incompatibility with legumes; or (iv) a gene consisting of an amino acid sequence in which 1 to 27 amino acid residues are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 4, which encodes a protein that induces symbiotic incompatibility with legumes.(7) (i) A gene consisting of a base sequence having 60% or more sequence identity with the base sequence represented by SEQ ID NO: 5, which induces symbiotic incompatibility with legumes; (ii) A gene consisting of a base sequence in which 1 to 83 bases are deleted, substituted, or added in the base sequence represented by SEQ ID NO: 5, which induces symbiotic incompatibility with legumes; (iii) A gene consisting of an amino acid sequence having 60% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, which encodes a protein that induces symbiotic incompatibility with legumes; or (iv) A gene consisting of an amino acid sequence in which 1 to 27 amino acid residues are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 6, which encodes a protein that induces symbiotic incompatibility with legumes. (8) (i) A gene consisting of a base sequence having 60% or more sequence identity with the base sequence represented by SEQ ID NO: 7, which induces symbiotic incompatibility with legumes; (ii) A gene consisting of a base sequence in which 1 to 81 bases are deleted, substituted, or added in the base sequence represented by SEQ ID NO: 7, which induces symbiotic incompatibility with legumes; (iii) A gene consisting of an amino acid sequence having 60% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 8, which encodes a protein that induces symbiotic incompatibility with legumes; or (iv) A gene consisting of an amino acid sequence in which 1 to 27 amino acid residues are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 8, which encodes a protein that induces symbiotic incompatibility with legumes.(9) (i) A gene consisting of a base sequence having 60% or more sequence identity with the base sequence represented by SEQ ID NO: 9, which induces symbiotic incompatibility with legumes; (ii) A gene consisting of a base sequence in which 1 to 39 bases are deleted, substituted, or added in the base sequence represented by SEQ ID NO: 9, which induces symbiotic incompatibility with legumes; (iii) A gene consisting of an amino acid sequence having 60% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 10, which encodes a protein that induces symbiotic incompatibility with legumes; or (iv) A gene consisting of an amino acid sequence in which 1 to 132 amino acid residues are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 10, which encodes a protein that induces symbiotic incompatibility with legumes.

[0055] The genes described in (5) and (6) may be genes that induce symbiotic incompatibility with legumes that express the GmNNL1 gene, the gene described in (7) may be a gene that induces symbiotic incompatibility with legumes that express the Rj2 gene, the gene described in (8) may be a gene that induces symbiotic incompatibility with legumes that express the Rfg1 gene, and the gene described in (9) may be a gene that induces symbiotic incompatibility with legumes that express the Rj4 gene.

[0056] In the genes (5) to (9), the sequence identity may be, for example, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0057] In the genes (5) to (8), the number of bases deleted, substituted, or added may be, for example, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2.

[0058] In the gene (9), the number of bases to be deleted, substituted, or added may be, for example, 1 to 390, 1 to 350, 1 to 300, 1 to 250, 1 to 200, 1 to 150, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2.

[0059] In the genes of (5) to (8), the number of amino acid residues to be deleted, substituted, or added may be, for example, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2.

[0060] In the gene (9), the number of amino acid residues to be deleted, substituted, or added may be, for example, 1 to 130, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2.

[0061] As used herein, "the function of an effector gene is deleted or reduced" means that the gene has been knocked out and / or its expression level has been reduced, and / or the function of the expression product of the gene has been deleted and / or reduced. The method for deleting or reducing the function of a gene may be performed by a method known to those skilled in the art. The deletion or reduction of the function of a gene may be due to a mutation in the gene or may be due to a mutation in the gene, or may be due to a mutation in the gene.

[0062] Whether or not the function of at least one effector gene that induces symbiotic incompatibility with legumes in rhizobia is deleted or reduced can be confirmed, for example, by inoculating the legume with the rhizobia, cultivating the legume, and then finding that the number of mature nodules formed on the roots is greater than the number of mature nodules formed by the same procedure except for inoculating a control carrying an effector gene instead of the rhizobia, for example, by 1.1 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 10 times or more, or 20 times or more. This indicates that the function of the effector gene in the rhizobia is deleted or reduced. Examples of situations in which it can be determined that the function of the effector gene of the rhizobia is missing or reduced include situations in which the effector gene-carrying control forms mature nodules, but the number of these is smaller than the number of mature nodules in the rhizobia, and situations in which the effector gene-carrying control forms nodule-like structures, but forms few or no mature nodules, and the number of these mature nodules is smaller than the number of mature nodules in the rhizobia.

[0063] When the loss or reduction of effector gene function is due to a mutation in the effector gene, the mutation is present in the transcription region (ORF or cDNA) of the effector gene. Examples of mutations include those that reduce the expression level of the effector gene (knockdown) when introduced, and those that eliminate the function of the effector gene (knockout). Methods for introducing mutations include, for example, mutation-based methods, genome editing techniques, insertion of exogenous factors, mutagen treatment, and introduction of loss-of-function genes. Mutation-based methods include isolating naturally mutated strains and inducing mutations. Methods for inserting exogenous factors include, for example, those using exogenous transposons. Mutagen treatments include, for example, treatment with chemical mutagens such as ethylmethanesulfonate (EMS), N-methyl-N-nitrosoguanidine (NTG), and nitrous acid, as well as treatment with physical mutagens such as gamma rays and heavy ion beams. Genome editing techniques include homologous recombination using vectors. Genome editing techniques include, for example, TALEN and CRISPR-Cas9. These methods can be used to modify the gene sequence of an effector gene by mutation, i.e., deletion, addition, or substitution of bases, thereby reducing the expression of the effector gene or deleting the function of the effector gene. Furthermore, by introducing a mutation into an effector gene, the function of the effector, which is its expression product, can be deleted or reduced. For example, a mutation in the effector gene can change the amino acids in the encoded effector, resulting in the deletion or reduction of the function of the original (wild-type) effector.

[0064] When the cause is something other than a mutation in the effector gene, for example, the expression level of the effector gene can be reduced (knocked down) by producing a transformant transformed with an antisense nucleic acid, siRNA, shRNA, miRNA, or other nucleic acid that induces RNA interference (RNAi), or a ribozyme, that targets the effector gene. When gene expression is reduced, the amount of its expression product (RNA or protein produced by expression) also decreases. Furthermore, for example, the transcription level of the effector gene can be reduced or translation can be inhibited by a mutation in a region (e.g., a regulatory region, an untranslated region) other than the transcription region (ORF or cDNA) of the effector gene that is involved in transcription or translation.

[0065] The loss or reduction of the function of the effector gene that induces symbiotic incompatibility with legumes in the target rhizobia is preferably due to the induction of a mutation in the effector gene or a mutation in the amino acid sequence of the effector gene. The mutation in the amino acid sequence of the effector gene may be performed by genome editing technology, preferably by a method utilizing homologous recombination using a vector.

[0066] The mutation in the effector gene is preferably caused by transposon insertion into the effector gene. The transposon may be an endogenous transposon. For example, when the effector gene inducing symbiotic incompatibility with a legume is the NopP gene or a gene shown in (5) to (8), the transposon insertion position may be any of the following positions (A) to (E) in these genes: (A) Between two consecutive bases in the base sequence corresponding to positions 715 to 815 of SEQ ID NO: 3. The position may be between two consecutive bases in the base sequence corresponding to positions 735 to 795 or positions 764 to 765 of SEQ ID NO: 3. (B) Between two consecutive bases in the base sequence corresponding to positions 505 to 605 of SEQ ID NO: 5. The position may be between two consecutive bases in the base sequence corresponding to positions 525 to 585 or positions 554 to 555 of SEQ ID NO: 5. (C) Between two consecutive bases in the base sequence corresponding to positions 105 to 205 of SEQ ID NO: 5. (D) Between two consecutive bases in a base sequence corresponding to positions 55 to 155 of SEQ ID NO: 5. The position may be between two consecutive bases in a base sequence corresponding to positions 75 to 135 or positions 103 to 104 of SEQ ID NO: 5. (E) Between two consecutive bases in a base sequence corresponding to positions 540 to 640 of SEQ ID NO: 5. The position may be between two consecutive bases in a base sequence corresponding to positions 560 to 620 or positions 592 to 593 of SEQ ID NO: 5.

[0067] Examples of transposon insertion sequences include ISBj11 (SEQ ID NO: 11), ISRj1 (SEQ ID NO: 12), and ISRj2 (SEQ ID NO: 13).

[0068] As rhizobia in which the function of at least one effector gene that induces symbiotic incompatibility with legumes is deleted or reduced due to transposon insertion, Bradyrhizobium ottawaense strain FY2-m1 (hereinafter also referred to as "FY2-m1 strain"), Bradyrhizobium ottawaense strain GMA461-m4 (hereinafter also referred to as "GMA461-m4 strain"), or Bradyrhizobium ottawaense strain OSA024 (hereinafter also referred to as "OSA024 strain") is preferred.

[0069] The FY2-m1 strain, GMA461-m4 strain, and OSA024 strain have excellent nodule occupancy rates and can infect legumes predominantly. For example, the nodule occupancy rates of the FY2-m1 strain, GMA461-m4 strain, and OSA024 strain on soybean expressing the GmNNL1 gene and the Rj2 gene can be 80% or more, 90% or more, or 95% or more. Furthermore, the FY2-m1 strain, GMA461-m4 strain, and OSA024 strain have high N ２ It has the ability to reduce O.

[0070] The FY2-m1 strain has been internationally deposited as NITE BP-04046 at the National Institute of Technology and Evaluation Patent Microorganisms Depositary, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, on December 14, 2023. In the FY2-m1 strain, ISBj2 (SEQ ID NO: 13) is inserted between the 554th and 555th bases in the base sequence of the NopP_122 gene (SEQ ID NO: 5).

[0071] The GMA461-m4 strain has been internationally deposited as NITE BP-04047 at the National Institute of Technology and Evaluation Patent Microorganisms Depositary, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, on December 14, 2023. In the GMA461-m4 strain, ISBj2 (SEQ ID NO: 13) is inserted between the 154th and 155th bases in the base sequence of the NopP_122 gene (SEQ ID NO: 5).

[0072] The OSA024 strain has been internationally deposited as NITE BP-04048 at the National Institute of Technology and Evaluation Patent Microorganisms Depositary, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, on December 14, 2023. In the OSA024 strain, ISBj11 (SEQ ID NO: 11) is inserted between the 764th and 765th bases in the base sequence of the NopP_6 gene (SEQ ID NO: 3).

[0073] Mutations in the amino acid sequence of the effector gene include mutations that cause the effector encoded by the effector gene to be a functionally deficient effector. A functionally deficient effector is preferably a protein encoded by the NopP gene or a gene shown in any of (5) to (8), in which the amino acid residue corresponding to position 60 in the amino acid sequence of NopP_110 (SEQ ID NO: 2) is substituted with leucine and the amino acid residue corresponding to position 271 in the amino acid sequence of NopP_110 (SEQ ID NO: 2) is substituted with phenylalanine. In this case, the NopP gene or a gene shown in any of (5) to (8) is more preferably the NopP_110 gene, the NopP_6 gene, or the NopP_122 gene, or a gene shown in any of (5) to (7), even more preferably the NopP_110 gene, the NopP_6 gene, or the NopP_122 gene, and particularly preferably the NopP_110 gene. However, in the above description of "an amino acid residue being substituted with phenylalanine," if the amino acid residue is originally phenylalanine, it does not need to be substituted. The same applies to leucine. In the amino acid sequence of NopP_110 (SEQ ID NO: 2), the amino acid sequence of NopP_6 (SEQ ID NO: 4), and the amino acid sequence of NopP_122 (SEQ ID NO: 6), the leucine or arginine at position 60, respectively, corresponds to the amino acid residue at position 60 in the amino acid sequence of NopP_110 (SEQ ID NO: 2), and the leucine or phenylalanine at position 271, respectively, corresponds to the amino acid residue at position 271 in the amino acid sequence of NopP_110 (SEQ ID NO: 2). That is, for example, the mutation in the amino acid sequence of the effector gene is preferably due to the substitution of leucine at position 271 with phenylalanine in the amino acid sequence of NopP_110.

[0074] As a rhizobia having a deletion or reduced function of at least one effector gene that induces symbiotic incompatibility with legumes due to a mutation in the amino acid sequence, a Bradyrhizobium ottawaense SG09 strain (hereinafter also referred to as "SG09 strain") is preferred, in which NopP is a protein in which the amino acid residue corresponding to position 60 of SEQ ID NO: 2 is substituted with leucine and the amino acid residue corresponding to position 271 is substituted with phenylalanine. In this case, the NopP is preferably a protein encoded by the NopP_110 gene, the NopP_6 gene, or the NopP_122 gene, or a gene shown in (5) to (7), more preferably the NopP_110 gene, the NopP_6 gene, or the NopP_122 gene, and particularly preferably the NopP_110 gene. That is, for example, as a rhizobia in which the function of at least one effector gene that induces symbiotic incompatibility with legumes is deleted or reduced due to a mutation in the amino acid sequence, a preferred strain is the SG09 strain in which NopP is a protein in which the leucine at position 271 of SEQ ID NO: 2 in NopP_110 is replaced with phenylalanine. The SG09 strain was deposited domestically as NITE BP-03361 at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, on January 5, 2021, and transferred to international deposit on November 8, 2021. The SG09 strain expresses the NopP6 gene.

[0075] Examples of methods for producing rhizobia lacking the function of an effector gene that induces symbiotic incompatibility with legumes include a production method (Method 1) comprising the step of isolating rhizobia lacking the function of the effector gene from mature root nodules of a legume; a production method (Method 2) comprising the steps of inoculating a legume that expresses an incompatibility gene with rhizobia that express an effector gene that induces symbiotic incompatibility with the incompatibility gene to form mature root nodules, and isolating rhizobia lacking the function of the effector gene from the mature root nodules; and a production method (Method 3) comprising the step of modifying the effector gene that induces symbiotic incompatibility with legumes in rhizobia, thereby causing the function of the effector gene to be deleted.

[0076] In Method 1, rhizobia in which the function of the effector gene is deleted may be isolated by, for example, collecting mature nodules from the roots of any legume, extracting DNA from the mature nodules by a conventional method, and analyzing the sequence of the effector gene to select rhizobia in which the function of the effector gene is deleted. The deletion of the function of the effector gene in the rhizobia may be due to spontaneous mutation.

[0077] In Method 1, it is preferable to select rhizobia in which the function of an effector gene is deleted, the deletion being due to transposon insertion into the effector gene.

[0078] Examples of sequence analysis of effector genes include PCR and DNA sequence analysis. The genome size of effector genes is often approximately 200 to 2,000 bp, while the genome size of transposons is often approximately 500 to 5,000 bp. Therefore, transposon insertion into effector genes can be confirmed by PCR by using a primer set designed to sandwich the transposon insertion site in the effector gene and comparing the length of the amplified fragment with that of an effector gene-carrying control. For example, when the effector gene is a NopP gene such as NopP_6 or NopP_122, the primer set may comprise a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 14 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 15.

[0079] When a transposon is inserted into an effector gene, the insertion position of the transposon in the effector gene and the sequence of the transposon can be confirmed by amplifying the effector gene region using a standard method and subjecting the amplified fragment to DNA sequencing analysis.

[0080] In Method 2, a legume plant expressing an incompatibility gene is inoculated with rhizobia expressing an effector gene that induces symbiotic incompatibility with the incompatibility gene, and in the step of forming mature nodules, mutations are induced in the rhizobia, thereby causing loss of function of the effector gene. In Method 2, loss of function of the effector gene is often caused by insertion of a transposon into the effector gene.

[0081] In Method 2, the step of isolating rhizobia in which the function of the effector gene is deleted from the nodules can be carried out in the same manner as in Method 1. In Method 2, it is preferable to select rhizobia in which the function of the effector gene is deleted by transposon insertion into the effector gene.

[0082] In methods 1 and 2, legume plants that express the Rj2 gene or the gene described in (2) are preferred, and legume plants that express the Rj2 gene are more preferred, from the viewpoint of making it easier to obtain rhizobia in which a transposon has been inserted into the effector gene of rhizobia.

[0083] In Methods 1 and 2, the genus, species, strain, etc. of the isolated rhizobia may be identified by phylogenetic analysis using the base sequence of the 16S-23S rDNA ITS region. The base sequence can be obtained by DNA sequence analysis.

[0084] In Method 3, examples of the method for recombining an effector gene that induces symbiotic incompatibility with a legume include a method for modifying the effector gene to a function-deficient type, and a method for knocking out the effector gene.

[0085] A preferred method for modifying an effector gene to be functionally defective is to modify the effector encoded by the effector gene to be a functionally defective effector. This method may, for example, include the steps of preparing a DNA fragment encoding the functionally defective effector and substituting the DNA fragment for the effector gene of a rhizobia. The step of preparing a DNA fragment encoding a functionally defective effector may be performed by using the genomic DNA of a rhizobia expressing the effector gene as a template, substituting the bases of the effector gene into the effector gene so that the effector is functionally defective, and amplifying the resulting DNA fragment. For example, if the effector gene is the NopP_110 gene (SEQ ID NO: 1), substituting the 811th cytosine with a thymine will render the encoded effector functionally defective. The step of substituting the DNA fragment for the effector gene of rhizobia may be carried out by a conventional method, for example, by introducing the DNA fragment into a suicide vector and substituting the DNA fragment for the effector gene of rhizobia through conjugation between E. coli transformed with the suicide vector and the rhizobia. Whether the effector gene has been modified to be functionally defective may be confirmed by DNA sequence analysis.

[0086] (Inoculation of rhizobia into legume plants) In the method of infecting legume plants with rhizobia according to this embodiment, the method and means for inoculating legume plants with rhizobia are not particularly limited as long as they can improve the nodule occupancy rate of the target rhizobia. For example, the rhizobia may be inoculated into seeds of a legume plant, or into the roots of a growing legume plant.

[0087] The mode of inoculating legumes with rhizobia may be to inoculate the surface of the legume seeds with rhizobia before sowing the seeds in soil, or to inoculate the soil near where the legumes are to be sown or have been sown. "Near the soil where the legumes are to be sown or have been sown" preferably means a position within 0.5 10 cm from the position where the legumes are to be sown or have been sown.

[0088] An example of inoculating the surface of legume seeds before sowing them in soil is to apply a composition for infecting legumes, which will be described later, to the surface of the legume seeds. In the above method, the composition may be in a liquid form, may contain a carrier, which will be described later, or may be a coating agent containing a carrier and a binder, which will be described later. In the above method, the seeds can be sown in soil after application.

[0089] Modes for inoculating legume plants into or near the soil where they have been sown include mode 1 in which a liquid composition for infecting legumes, described below, is sprayed or dripped onto the seeds and the soil near the seeds at the same time as the legumes are sown, mode 2 in which a composition for infecting legumes containing a carrier, described below, is sprayed near the soil where the legumes have been sown, and mode 3 in which a liquid composition for infecting legumes, described below, is sprayed near the soil where the legumes have been sown. In these modes, the rhizobia may be inoculated into the seeds of the legumes or into the roots of the legumes, with inoculation into the seeds of the legumes being preferred.

[0090] In Aspect 2, the application is preferably carried out within 24 hours, more preferably within 8 hours, and particularly preferably within 30 minutes from the time of sowing the seeds of the legume into the soil. In Aspect 2, the carrier is preferably peat moss.

[0091] In Aspect 3, the spraying is preferably carried out within 8 hours, more preferably within 30 minutes, from the time of sowing the seeds of the legume into the soil. In Aspect 3, the soil may be in the form of furrows.

[0092] The soil is not particularly limited as long as it allows legumes to grow, and may be, for example, soil designated as culture soil, fertilizer, seedling raising soil, or seedling raising soil, or soil from mountains or fields that has not been treated in any way may be used as is.

[0093] The inoculation of rhizobia into legumes can be set appropriately depending on the type of legume and rhizobia. For example, when the legume is soybean and the rhizobia is Bradyrhizobium bacteria, 1 to 5 × 10 ５ cfu (colony forming units), and 5 x 10 to 5 x 10 ４ It may be cfu.

[0094] [Composition for infecting legume plants] The composition for infecting legume plants according to this embodiment contains one or more species of rhizobia lacking the function of an effector gene that induces symbiotic incompatibility with legume plants, as used in the [method for infecting legume plants with rhizobia]. Furthermore, the composition may contain, in addition to rhizobia, a bacterial species that promotes plant growth, for example. The composition may also be a material for infecting legume plants with rhizobia.

[0095] The composition for infecting legumes may be a material in liquid form, a material in the form of a carrier carrying rhizobia, or a material in which the surface of seeds is coated with rhizobia.

[0096] Among the above compositions, the liquid material is a material in which the target rhizobia are cultured and concentrated, and the liquid medium is water. The liquid material may contain, for example, trace metal ions, bacterial extracellular polymers, sugars, etc. Furthermore, the liquid material may contain other bacterial species in addition to the target rhizobia. The liquid material may be applied to seeds before sowing in soil, or may be sprayed in the vicinity of the sown soil.

[0097] When the composition is a liquid material, the content of the root nodule bacteria in the liquid medium is 1 x 10 ５ ~1 x 10 １０ cfu / ml, and 5 x 10 ８ ~5 x 10 ９ It may be cfu / ml.

[0098] Among the above compositions, the material in the form of rhizobia supported on a carrier is a material in which the target rhizobia is supported on an agriculturally acceptable carrier. The carrier may be any material capable of supporting rhizobia, including peat moss, zeolite, vermiculite, montmorillonite, coco peat, perlite, Akadama soil, Kanuma soil, Hyuga soil, and leaf mold, with peat moss or zeolite being preferred. The composition may contain one type of carrier or two or more types of carriers. The material in the form of rhizobia supported on a carrier may be used by spraying or mixing with soil near where the seeds are sown. Alternatively, the material in the form of rhizobia supported on a carrier may be used by applying or dusting seeds before sowing them in the soil.

[0099] When the composition is a material in the form of a carrier (peat moss) carrying rhizobia, the content of the rhizobia carried on the carrier is 1 x 10 ５ ~1 x 10 １０ cfu / g, and 5 x 10 ８ ~5 x 10 ９ It may also be cfu / g.

[0100] Among the above compositions, the material in the form of coating the surface of a seed may contain the target rhizobia, a carrier, and a binder. The carrier may be any material capable of supporting the rhizobia, such as peat moss, zeolite, vermiculite, montmorillonite, cocopeat, perlite, Akadama soil, Kanuma soil, Hyuga soil, and leaf mold. Examples of binders include carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, starch, polyethylene glycol, polyvinyl alcohol, and gelatin. Since the material in the form of coating the surface of a seed already contains the target rhizobia in the coating agent, the target rhizobia can be inoculated into the seeds by sowing the seeds in soil.

[0101] When the composition is in the form of a material coated on the surface of a seed, the content of the rhizobia coated per seed is 1 to 5 x 10 ５ cfu / unit, or 5 x 10 to 5 x 10 ４ It may be cfu / unit.

[0102] The composition may further contain a surfactant, a preservative, a dispersant, an antioxidant, etc. The composition may also be formulated into a dosage form such as a liquid, a suspension, an emulsion, or a paste. The formulation can be carried out by a conventional method. A formulation of the coating composition can be called a coating agent.

[0103] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0104] Example 1-1 Creation of Rj2 / GmNNL1 gene integrated soybean lines The soybean varieties "Bonminori" (JP28493, Norin No. 26) and "WC108 Black Bean" (JP27584) were crossed, and lines homozygously carrying the Rj2 / GmNNL1 gene were selected from the F2 seeds of the cross progeny, and F3 seeds of these progeny lines were obtained. The specific procedure is as follows. First, the soybean varieties "Bonminori" and "WC108 Black Bean" were cultivated so that they had the same flowering period, and crossed by pollinating emasculated buds from the maternal side with pollen from the paternal side. DNA was extracted from the cotyledons of the resulting hybrid seeds, and the Rj2 and GmNNL1 gene markers were amplified by PCR using Primer Set 1 (forward primer: SEQ ID NO: 24, reverse primer: SEQ ID NO: 25) and Primer Set 2 (forward primer: SEQ ID NO: 26, reverse primer: SEQ ID NO: 27), respectively. The amplified fragments were then subjected to agarose gel electrophoresis, and F1 seeds with heterozygous Rj2 and GmNNL1 genes were selected based on their migration patterns. The F1 seeds were sown and grown, and DNA was extracted from the cotyledons of the F2 seeds harvested from the resulting F1 plants. The Rj2 and GmNNL1 gene markers were amplified in the same manner as in the PCR method described above. The amplified fragments were subjected to agarose gel electrophoresis, and F2 seed lines with homozygous Rj2 and GmNNL1 genes were selected based on their migration patterns (Figure 1). The selected F2 seeds were sown and grown, and F3 seeds were harvested from the resulting F2 plants to obtain the Rj2 / GmNNL1 soybean line. Hereinafter, soybeans homozygous for the Rj2 / GmNNL1 gene are also referred to as "Rj2 / GmNNL1 soybeans."

[0105] Figure 1 shows an agarose gel electrophoresis image of PCR-amplified fragments of the Rj2 and GmNNL1 gene marker regions of cotyledon DNA from F2 seeds. The genotypes of Rj2 and rj2 can be distinguished by differences in band length. Similarly, the genotypes of GmNNL1 and Gmnnl1 can also be distinguished by differences in band length. Of the eight progeny individuals, individual number 8 showed only bands for the Rj2 and GmNNL1 genotypes, confirming that it is a line that has Rj2 and GmNNL1 homozygously.

[0106] Example 1-2 [Creation of nopP gene function-loss strain] A nopP gene function-loss strain (hereinafter also referred to as "ΔnopP strain") was created by the method described below, and a strain in which a transposon had been inserted into the nopP gene was selected by nopP gene sequence analysis as described below.

[0107] (nopP Gene Sequence Analysis) After extracting DNA from the strains, the nopP gene region was amplified by PCR using Primer Set 1 (forward primer: SEQ ID NO: 14, reverse primer: SEQ ID NO: 15), and the amplified fragment was subjected to agarose gel electrophoresis. Strains in which the length of the amplified fragment was shifted toward higher molecular weights compared to strains carrying the nopP gene were selected as strains in which a transposon had been inserted into the nopP gene. The nopP gene-carrying strains used were Bradyrhizobium japonicum USDA6 strain (MAFF number: 211646), Bradyrhizobium diazoefficiens USDA110 strain (JCM number: 10833), and Bradyrhizobium diazoefficiens USDA122 strain (BCRC number: 13533). Furthermore, the amplified fragments of the selected strains were subjected to the Sanger method to determine the DNA sequence, and it was confirmed that the transposon had been inserted.

[0108] (Creation of Bradyrhizobium ottawaense OSA024 strain) Soil was collected from a soybean field in Osaka Prefecture in 2022, and soybeans (variety: Enrei) were cultivated in a greenhouse using pots containing the soil, and mature nodules were collected from the soybean roots. Rhizobium carrying the Bradyrhizobium ottawaense nosZ gene was selected by PCR analysis of each mature nodule, and then pure isolation was carried out on HM agar plate medium. Figure 2(C) shows the positional relationship between the nopP gene region and the forward and reverse primers. Rhizobium carrying the Bradyrhizobium ottawaense nosZ gene is known to be highly resistant to the greenhouse gas nitrous oxide (N ２O) to nitrogen. The isolated rhizobia strains were subjected to phylogenetic analysis based on the nucleotide sequence of the 16S-23S rDNA ITS region to select strains with nucleotide sequence identity of 99% or more with the Bradyrhizobium ottawaense SG09 strain, and the above-described nopP gene sequence analysis was used to select strains with a transposon inserted into the nopP gene. The selected strain was designated the Bradyrhizobium ottawaense OSA024 strain, which exhibits nopP gene loss of function. Figure 2(A) shows the transposon insertion site in the nopP_6 gene obtained by Sanger analysis. The transposon insertion sequence was ISBj11 (SEQ ID NO: 11). The results of electrophoresis of the amplified fragment of the nopP gene region are shown in Figure 2(B).

[0109] (Creation of Bradyrhizobium ottawaense FY2-m1 and GMA461-m4 strains) Mature nodules were collected from soybean roots grown in a field in Gunma Prefecture in 2021 and 2022. Rhizobium harboring the Bradyrhizobium ottawaense nosZ gene was selected by PCR analysis of each mature nodule, followed by pure isolation on HM agar plates. Phylogenetic analysis was performed on the isolated rhizobium strains based on the nucleotide sequence of the 16S-23S rDNA ITS region, and strains with nucleotide sequence identity of 99% or more to the Bradyrhizobium ottawaense SG09 strain were selected and designated Bradyrhizobium ottawaense FY2 and Bradyrhizobium ottawaense GMA461, respectively. FY2 and GMA461 strains express NopP_122.

[0110] Rhizobium bacteria were isolated from large mature nodules formed by inoculating Bradyrhizobium ottawaense FY2 strain and GMA461 strain onto soybean (cultivar: Hardee) carrying the Rj2 incompatibility gene. The isolated strains were subjected to the above-described nopP gene sequence analysis, and strains with transposon insertion in the nopP gene were selected. Four Bradyrhizobium ottawaense FY2 strains were isolated from the mature nodules, and transposon insertion in the nopP gene was confirmed in three of these strains. One of these strains was selected as a representative strain and designated Bradyrhizobium ottawaense FY2-m1 strain. Four strains of Bradyrhizobium ottawaense GMA461 were isolated from the mature nodules, and transposon insertion into the nopP gene was confirmed in two of these strains. One of these strains was selected as a representative strain and designated Bradyrhizobium ottawaense GMA461-m4. In the nopP gene sequence analysis described above, the transposon insertion position in the nopP gene obtained by the Sanger method is shown in Figure 3(A), and the results of electrophoresis are shown in Figure 3(B). In all strains, the transposon insertion sequence was ISRj2 (SEQ ID NO: 13).

[0111] (Creation of Bradyrhizobium diazoefficiens W3-1a and W9-1a Strains) Bradyrhizobium diazoefficiens USDA122 strain was inoculated onto soybean (cultivar: Hardee) having the Rj2 incompatibility gene, and rhizobia were isolated from the large mature nodules formed. The isolated strains were subjected to the above-described nopP gene sequence analysis, and strains in which a transposon had been inserted into the nopP gene were selected. Eight strains were isolated from the mature nodules, and transposon insertion into the nopP gene was confirmed in two of these strains, and these strains were designated Bradyrhizobium diazoefficiens W3-1a and W9-1a, respectively. Figure 3(A) shows the transposon insertion position in the nopP gene obtained by the Sanger method in the above-described nopP gene sequence analysis, and Figure 3(B) shows the results of electrophoresis. In the W3-1a strain, the transposon insertion sequence was ISRj2 (SEQ ID NO: 13), and in the W9-1a strain, the transposon insertion sequence was ISRj1 (SEQ ID NO: 12).

[0112] (Creation of a genetically modified strain of Bradyrhizobium ottawaense SG09 strain) The nopP gene present on the genome of the Bradyrhizobium ottawaense SG09 strain was modified to a base sequence that can avoid symbiotic incompatibility with GmNNL1- and Rj2-accumulating soybeans. The specific method is as follows. First, using the genomic DNA of the Bradyrhizobium diazoefficiens USDA110 strain as a template, a nopP gene fragment (hereinafter referred to as "nopP") in which base 811 in the nopP gene base sequence (SEQ ID NO: 1) was modified from cytosine to thymine was generated by overlap PCR. ＬＦThis was then inserted into the SmaI restriction enzyme site of the suicide vector pK18mobsacB-Ω (Wasai-Hara et al., unpublished) to obtain the plasmid pMS192. Next, a mixture of culture fluids of E. coli containing the above-mentioned plasmid pMS192, E. coli containing the helper plasmid pRK2013, and Bradyrhizobium ottawaense SG09 strain was spotted onto an HM agar medium and incubated at 30°C for 3 days. The grown cells were suspended in sterile distilled water and plated on an HM agar medium containing spectinomycin (100 μg / ml), streptomycin (100 μg / ml), and polymyxin B (50 μg / ml), and the grown rhizobia strain was obtained. The obtained rhizobia strain was cultured in HM liquid medium for 5 days, and then spread onto HM agar medium containing 10% sucrose. After culturing for 10 days, multiple colonies emerged and were obtained. DNA was extracted from the obtained strain, and the base sequence of the nopP gene region was confirmed by the Sanger method. ＬＦ A strain having the formula Bradyrhizobium ottawaense SG09nopP was selected. ＬＦ It was decided.

[0113] Comparative Example 1 In the following examples, Bradyrhizobium japonicum USDA6 strain, Bradyrhizobium diazoefficiens USDA110 strain, and Bradyrhizobium diazoefficiens USDA122 strain were used as strains carrying the nopP gene.

[0114] [Inoculation of nopP gene function-loss strains into Rj2 / GmNNL1 gene-integrated soybeans] <Example 2> 6.7 × 10 ２The microbial material was prepared by suspending the soybeans in sterilized water to a concentration of 150 mL / mL. 150 mL of the microbial material was added to Leonardo jar pots containing sterilized vermiculite, and chlorine gas-fumigated Rj2 / GmNNL1 soybeans were sown at two seeds per pot. Cultivation was carried out in a climate chamber at 25°C under conditions of 16 hours of light and 8 hours of darkness. Four days after sowing, the soybeans were thinned out, leaving only one with good germination, and then cultivated for three weeks. The pots were periodically supplied with nitrogen-free hydroponic solution.

[0115] Comparative Example 2 The same procedure as in Example 2 was carried out, except that the nopP gene-containing strains GMA461, FY2, and USDA122 were used instead of the nopP gene function-losing strain.

[0116] Figure 4 shows photographs of soybean roots cultivated in Example 2 and Comparative Example 2, and a graph showing the number of mature nodules formed. Figure 4(A) shows the GMA461 and GMA461-m4 strains, Figure 4(B) shows the FY2 and FY2-m1 strains, and Figure 4(C) shows the USDA122, W3-1a, and W9-1a strains. The vertical axes of Figures 4(A) to 4(C) represent the number of mature nodules. As shown in Figure 4(A), multiple mature nodules were formed in roots inoculated with the GMA461-m4 strain, a nopP gene function-deficient strain, whereas nodule-like structures were observed in roots inoculated with the GMA461 strain, but no mature nodule formation was confirmed. Similarly, Figure 4(B) shows that multiple mature nodules were formed in roots inoculated with the FY2-m1 strain, a nopP gene function-deficient strain, whereas no mature nodule formation was confirmed in roots inoculated with the FY2 strain. In Figure 4(C), multiple mature nodules were observed in the roots inoculated with the W3-1a and W9-1a strains, but no mature nodule formation was observed in the roots inoculated with the USDA122 strain. These results demonstrate that the nopP gene function-deficient strains GMA461-m4, FY2-m1, W3-1a, and W9-1a strains can bypass incompatibility, infect Rj2 / GmNNL1 soybean, and form mature nodules.

[0117] Example 3 The nopP gene function-deficient strains GMA461-m4, FY2-m1, OSA024, W3-1a, and W9-1a were each suspended in sterilized water. The nopP gene-carrying strains USDA6, USDA110, and USDA122 were each suspended in sterilized water. Any one of the nopP gene function-deficient strains and all three nopP gene-carrying strains were mixed in a 1:1:1:1 ratio to obtain a total cell count of 6.7 x 10 ２ A microbial material adjusted to 1000 cells / mL was prepared. 150 mL of the microbial material prepared above was added to Leonardo jar pots containing sterilized vermiculite, and chlorine gas-fumigated Rj2 / GmNNL1 soybeans were sown at two seeds per pot. Cultivation was carried out in an artificial climate chamber at 25°C under conditions of 16 hours of light and 8 hours of darkness. Four days after sowing, the soybeans were thinned out, leaving only one with good germination, and then cultivated for three weeks. The pots were periodically supplied with nitrogen-free hydroponic solution.

[0118] After cultivation, the number of mature nodules formed on the roots was counted, and the mature nodules were collected and immersed in 0.5% sodium hypochlorite solution for 3 minutes to sterilize the nodule surface. Each mature nodule was disrupted and DNA extracted from each nodule. The nopP gene region was amplified by PCR using the forward primer represented by SEQ ID NO: 14 and the reverse primer represented by SEQ ID NO: 15. The PCR-amplified fragment was cleaved with the restriction enzymes AluI and PstI and subjected to agarose gel electrophoresis. The strains occupying the mature nodules were identified from the band patterns of the restriction enzyme-cleaved PCR-amplified fragments. Figure 5 shows an example of the band patterns of the restriction enzyme fragments of each strain. The USDA6, USDA110, and USDA122 strains exhibited the band patterns shown in the corresponding lanes in the figure, whereas the nopP gene function-loss strains GMA461-m4, FY2-m1, W3-1a, W9-1a, and OSA024 strains exhibited different band patterns depending on the endogenous transposon insertion site in the nopP gene sequence.

[0119] Comparative Example 3 The same procedure as in Example 3 was carried out, except that a soybean variety not carrying an incompatibility gene (Akutashirazu) was used instead of the Rj2 / GmNNL1 soybean.

[0120] Table 1 shows the percentage of mature nodules occupied by each strain in mature nodules of Rj2 / GmNNL1 soybeans and Akutashiraji soybeans formed by simultaneous inoculation of the nopP gene function-deficient strain GMA461-m4 with USDA6, USDA110, and USDA122 strains in Example 3 and Comparative Example 3. In Table 1, "mix" represents the percentage of mature nodules infected with multiple rhizobia. While the percentage of mature nodules occupied by the GMA461-m4 strain in Akutashiraji soybeans, which do not possess incompatibility genes, was 1.6%, the percentage of mature nodules occupied by the GMA461-m4 strain in Rj2 / GmNNL1 soybeans was 95.8%.

[0121]

[0122] Table 2 shows the percentage of mature nodules occupied by each strain in mature nodules of Rj2 / GmNNL1 soybeans and Akutashiraji soybeans formed by simultaneous inoculation of the nopP gene function-deficient strain FY2-m1 with the USDA6, USDA110, and USDA122 strains in Example 3 and Comparative Example 3. In Table 2, "mix" represents the percentage of mature nodules infected with multiple rhizobia. While the percentage of mature nodules occupied by the FY2-m1 strain in Akutashiraji soybeans, which do not possess incompatibility genes, was 0.0%, the percentage of mature nodules occupied by the FY2-m1 strain in Rj2 / GmNNL1 soybeans was 96.9%.

[0123]

[0124] Table 3 shows the percentage of mature nodules occupied by each strain in mature nodules of Rj2 / GmNNL1 soybeans and Akutashiraji soybeans formed by simultaneous inoculation of the nopP gene function-deficient strain OSA024 with the USDA6, USDA110, and USDA122 strains in Example 3 and Comparative Example 3. In Table 3, "mix" represents the percentage of mature nodules infected with multiple rhizobia. While the percentage of mature nodules occupied by the OSA024 strain in Akutashiraji soybeans, which do not possess incompatibility genes, was 1.1%, the percentage of mature nodules occupied by the OSA024 strain in Rj2 / GmNNL1 soybeans was 91.1%.

[0125]

[0126] Table 4 shows the percentage of mature nodules occupied by each strain in Rj2 / GmNNL1 soybeans and Akutashiraji soybeans formed by simultaneous inoculation of the nopP gene function-deficient strain W3-1a with the USDA6, USDA110, and USDA122 strains in Example 3 and Comparative Example 3. In Table 4, "mix" represents the percentage of mature nodules infected with multiple rhizobia. While the percentage of mature nodules occupied by the W3-1a strain in Akutashiraji soybeans, which do not possess incompatibility genes, was 2.1%, the percentage of mature nodules occupied by the W3-1a strain in Rj2 / GmNNL1 soybeans was 38.2%. The W3-1a strain was created by inducing mutations in the USDA122 strain, and it was confirmed that the proportion of mature nodules occupied by the W3-1a strain was improved compared to 0.0% for the USDA122 strain.

[0127]

[0128] Table 5 shows the percentage of mature nodules occupied by each strain in Rj2 / GmNNL1 soybeans and Akutashiraji soybeans formed by simultaneous inoculation of the nopP gene function-deficient strain W9-1a with the USDA6, USDA110, and USDA122 strains in Example 3 and Comparative Example 3. In Table 5, "mix" represents the percentage of mature nodules infected with multiple rhizobia. While the percentage of mature nodules occupied by the W9-1a strain in Akutashiraji soybeans, which do not possess incompatibility genes, was 1.7%, the percentage of mature nodules occupied by the W9-1a strain in Rj2 / GmNNL1 soybeans was 15.4%. The W9-1a strain was created by inducing mutations in the USDA122 strain, and it was confirmed that the proportion of mature nodules occupied by the W9-1a strain was improved compared to 0.0% for the USDA122 strain.

[0129]

[0130] Figure 6 shows the total number of mature nodules (gray + black) that grew on Akutashirazu and the number of mature nodules occupied by the nopP gene function-deficient strain (ΔnopP) (black) in Comparative Example 3. In Figure 6, "a," "b," and "ab" are labels used in the Tukey HSD test, and the significance level between different labels is 0.05 or less. As shown in Tables 1 to 5, the nodule occupancy rate of the ΔnopP strain remained at 0.0 to 2.1% for all nopP gene function-deficient strains on Akutashirazu. In contrast, Figure 7 shows the total number of mature nodules (gray + black) that grew on Rj2 / GmNNL1 soybean in Example 3 and the number of mature nodules occupied by the nopP gene function-deficient strain (black). In Figure 7, "a," "b," "ab," and "c" are labels in the Tukey HSD test, and the significance level between different labels is 0.05 or less. Among the nopP gene function-loss strains, GMA461-m4, FY2-m1, and OSA024 had a large proportion of mature nodules occupied by these strains.

[0131] The above data indicate that the nopP gene function-deficient strains obtained in this invention as rhizobia that avoid incompatibility with Rj2 / GmNNL1 soybean, particularly FY2-m1, GMA461-m4, and OSA024 strains, have extremely high nodule formation and nodule occupancy abilities under competitive inoculation conditions with multiple other rhizobia.

[0132] Example 4: SG09 strain and a recombinant strain of SG09 strain with modified nopP amino acid sequence (SG09nopP ＬＦ strains) were mixed in a ratio of approximately 1:1, and 1.0 × 10 4 An inoculum was prepared by diluting the solution with sterilized water to 1000 cells / mL. After chlorine gas fumigation, pre-germinated Rj2 / GmNNL1 soybeans were transplanted into Leonardo jar pots containing sterilized vermiculite, two plants per pot, and 1 mL of the inoculum prepared above was inoculated into each pot. Cultivation was carried out in a climate chamber at 25°C under conditions of 16 hours light / 8 hours dark for 3 weeks. The pots were periodically supplied with nitrogen-free hydroponic solution.

[0133] After cultivation, the number of mature nodules formed on the roots was counted, and the mature nodules were collected and immersed in a 0.5% sodium hypochlorite solution for 3 minutes to sterilize the nodule surface. Each mature nodule was disrupted to extract DNA. The nopP gene region was amplified by PCR using the forward primer represented by SEQ ID NO: 14 and the reverse primer represented by SEQ ID NO: 15. The PCR-amplified fragment was cleaved with the restriction enzyme AluI and subjected to agarose gel electrophoresis. The strains occupying the nodules were identified from the band patterns of the restriction enzyme-cleaved PCR-amplified fragments. Figure 8(A) shows an example of the band patterns of the restriction enzyme fragments of each strain. The SG09 strain and SG09nopP ＬＦ Each strain exhibited a different band pattern as shown in the corresponding lane in the figure.

[0134] Comparative Example 4: Instead of Rj2 / GmNNL1 soybean, a soybean variety (Yukihomare R) not carrying an incompatibility gene was used, and the same procedure as in Example 3 was carried out. ＬＦ The abundance ratio of the strains was investigated in the same manner as above, using DNA extracted from each colony formed on the HM agar medium as a template.

[0135] FIG. 8(B) shows the SG09 strain and SG09nopP in Example 4 and Comparative Example 4. ＬＦ SG09 strain and SG09nopP in the inoculum when the strains were simultaneously inoculated ＬＦ The graph shows the percentage of the SG09 strain in the inoculum, as well as the percentage of mature nodules occupied by each strain in the mature nodules of Rj2 / GmNNL1 soybean and Yukihomare R soybean. ＬＦ In Example 4 and Comparative Example 4, the SG09nopP strain in Yukihomare R, which does not have an incompatibility gene, showed a 45.0%. ＬＦ The percentage of mature nodules occupied by the strain was 58.0%, whereas SG09nopP in Rj2 / GmNNL1 soybean ＬＦ The percentage of mature nodules occupied by the strain was 95.0%. ＬＦThe strain was shown to have the ability to circumvent incompatibility caused by the Rj2 and GmNNL1 genes and increase nodule occupancy on soybean.

[0136] Example 5 In the Kashimadai field of Tohoku University, Rj2 / GmNNL1 soybeans were soybean cultured with peat moss materials containing the SG09 strain, the GMA461 strain, and the GMA461-m4 strain for 10 minutes. １０ The inoculation was carried out at a concentration of 100 colony-forming units / seed (June 13, 2023). Approximately one month later (July 27, 2023), the percentage of mature nodules occupied by the inoculated bacteria (hereinafter also referred to as "nodule occupancy") was quantified by PCR analysis detecting the Bradyrhizobium ottawaense nosZ gene. Furthermore, approximately three months later (September 25), the N released from the field soil during the soybean ripening period was analyzed. ２ O emission amount (N ２ The O flux was measured. ２ The amount of O released was measured using a mid-infrared laser gas monitor.

[0137] Figure 9(A) shows the nodule occupancy rates of SG09, GMA461, and GMA461-m4 strains in the field inoculation test, and Figure 9(B) shows the N nodule occupancy rates of soybean cultivated fields inoculated with each strain. ２ O flux (μgNm －２ h －１ ) are shown. In Figure 9, "a" and "b" are labels in the Tukey HSD test, and the significance level between different labels is 0.05 or less. As shown in Figure 9(A), the nodule occupancy rate of the GMA461 strain was 0%, but it reached 62% for the nopP gene function-deficient strain GMA461-m4, indicating a significant increase in the nodule occupancy rate of the GMA461-m4 strain. Furthermore, as shown in Figure 9(B), the N of soybeans inoculated with the GMA461-m4 strain taken from field soil during the soybean ripening period about 3 months later (September 25th) was ２ The O flux was significantly reduced by more than 60% compared to the GMA461 strain-inoculated soybean, and the N flux was clearly reduced by the improved nodule occupancy rate of the GMA461-m4 strain. ２ The effect of reducing O generation was observed.

[0138] N in the soybean rhizosphere ２Bradyrhizobium ottawaense, including the SG09 strain, has several times higher O reduction activity than Bradyrhizobium diazoefficiens (Wasai-Hara et al. 2023). The Bradyrhizobium ottawaense FY2-m1, GMA461-m4, and OSA024 strains obtained in the present invention have the same N reduction activity as the Bradyrhizobium ottawaense SG09 strain. ２ It has the ability to reduce greenhouse gases N ２ From the viewpoint of O reduction, FY2-m1, GMA461-m4, OSA024 strain, and SG09nopP ＬＦ The strain is said to be excellent.

[0139] Example 6 The nopP gene function-deficient strains GMA461-m4, FY2-m1, and OSA024 were each suspended in sterilized water. The nopP gene-carrying strains USDA6, USDA110, and USDA122 were each suspended in sterilized water. Any one of the nopP gene function-deficient strains and all three nopP gene-carrying strains were mixed in a ratio of 1:1:1:1, resulting in a total cell count of 6.7 x 10 ２ A microbial material adjusted to 1000 cells / mL was prepared. 150 mL of the microbial material prepared above was added to Leonardo jar pots containing sterilized vermiculite, and chlorine gas-fumigated Rj2 / GmNNL1 soybeans were sown at two seeds per pot. Cultivation was carried out in an artificial climate chamber at 25°C under conditions of 16 hours of light and 8 hours of darkness. Four days after sowing, the soybeans were thinned out to one with good germination per Leonardo jar pot, and then cultivated for five weeks. The pots were periodically supplied with nitrogen-free hydroponic solution.

[0140] After the 5-week cultivation period, soybeans were harvested from the pots, the above-ground parts were excised, and the roots were washed with tap water. For the nodule aging treatment, soil collected on May 21, 2024, from the Kashimadai field at Tohoku University was used. Specifically, soybean roots were placed in a 100 mL glass vial, and 30 mL of the soil was added to bury the roots. Then, 10 mL of distilled water was added and the vial was incubated in the dark at 25°C in an open state to allow nodule aging to proceed. To compensate for the water that evaporated during incubation, the vial was weighed once a week, and distilled water was added to compensate for the weight loss. One, two, and three weeks after the start of the aging treatment, the vial was sealed under atmospheric conditions for approximately 3 to 7 hours, and the gas phase was sampled. The N in the gas phase before and after sealing was measured using an ECD gas chromatograph (Shimazu, G-C2014). ２ The O concentration was measured and the N concentration was measured under atmospheric conditions. ２ O emission amount (N ２ O flux) (nmol h －１ plant －１ ) was sought.

[0141] Comparative Example 5 The same procedures as in Example 6 were carried out, except that rj2 / Gmnnl1 soybean (a line selected from the same progeny line as the Rj2 / GmNNL1 soybean) not carrying the incompatibility gene was used instead of Rj2 / GmNNL1 soybean.

[0142] Table 6 shows the percentage of mature nodules occupied by each strain in mature nodules of Rj2 / GmNNL1 soybeans and rj2 / Gmnnl1 soybeans formed by simultaneous inoculation of the nopP gene function-deficient strain GMA461-m4 with the USDA6, USDA110, and USDA122 strains in Example 6 and Comparative Example 5. In Table 6, "mix" represents the percentage of mature nodules infected with multiple rhizobia. The percentage of mature nodules occupied by the GMA461-m4 strain in rj2 / Gmnnl1 soybeans not carrying an incompatibility gene was 54.7%, while the percentage of mature nodules occupied by the GMA461-m4 strain in Rj2 / GmNNL1 soybeans was 92.1%.

[0143]

[0144] Table 7 shows the percentage of mature nodules occupied by each strain in mature nodules of Rj2 / GmNNL1 soybeans and rj2 / Gmnnl1 soybeans formed by simultaneous inoculation of the nopP gene function-deficient strain FY2-m1 with the USDA6, USDA110, and USDA122 strains in Example 6 and Comparative Example 5. In Table 7, "mix" represents the percentage of mature nodules infected with multiple rhizobia. The percentage of mature nodules occupied by the FY2-m1 strain in rj2 / Gmnnl1 soybeans not carrying an incompatibility gene was 56.1%, while the percentage of mature nodules occupied by the FY2-m1 strain in Rj2 / GmNNL1 soybeans was 95.3%.

[0145]

[0146] Table 8 shows the percentage of mature nodules occupied by each strain in mature nodules of Rj2 / GmNNL1 soybeans and rj2 / Gmnnl1 soybeans formed by simultaneous inoculation of the nopP gene function-deficient strain OSA024 with the USDA6, USDA110, and USDA122 strains in Example 6 and Comparative Example 5. In Table 8, "mix" represents the percentage of mature nodules infected with multiple rhizobia. The percentage of mature nodules occupied by the OSA024 strain in rj2 / Gmnnl1 soybeans not carrying an incompatibility gene was 27.9%, while the percentage of mature nodules occupied by the OSA024 strain in Rj2 / GmNNL1 soybeans was 97.4%.

[0147]

[0148] Figure 10 shows the total number of mature nodules (white + black) that formed on rj2 / Gmnnl1 and the number of mature nodules occupied by the nopP gene function-deficient strain (ΔnopP) (black) in Comparative Example 5. No significant differences were observed in the number of nodules among the test plots in Figure 10. As shown in Tables 6 to 8, in rj2 / Gmnnl1 soybeans, the nodule occupancy rate of the ΔnopP strain was only 27.9 to 56.1% for all nopP gene function-deficient strains. In contrast, Figure 11 shows the total number of mature nodules (white + black) that formed on Rj2 / GmNNL1 soybeans in Example 6 and the number of mature nodules occupied by the nopP gene function-deficient strain (black). No significant differences were observed in the number of nodules among the test plots in Figure 11. As shown in Tables 6 to 8, the nodule occupancy rate of the ΔnopP strain exceeded 90% for all the nopP gene function-deficient strains.

[0149] In FIG. 12, the solid line indicates the N produced during senescence of Rj2 / GmNNL1 soybeans, as measured in Example 6. ２ The graph shown by the dotted line indicates the amount of N released during senescence of rj2 / Gmnnl1 soybeans, as measured in Comparative Example 5. ２ In Fig. 12, each data point is expressed as mean value ± standard error. The asterisks in Fig. 12 indicate the amount of N released at 1 week, 2 weeks, and 3 weeks after aging treatment. ２ This label indicates that the significance level of the Student's t-test between Rj2 / GmNNL1 soybean and rj2 / Gmnnl1 soybean is less than 0.05. Among the nopP gene function-loss strains, GMA461-m4 and FY2-m1 showed a difference in the amount of O released from Rj2 / GmNNL1 soybean at 3 weeks after the start of senescence. ２ The amount of O released by OSA024 was significantly reduced compared to that of rj2 / Gmnnl1 soybeans. Furthermore, at 2 and 3 weeks after the start of senescence, OSA024 significantly reduced the amount of O released by Rj2 / GmNNL1 soybeans. ２ The amount of O released was significantly reduced compared to rj2 / Gmnnl1 soybean.

[0150] The above data indicate that FY2-m1, GMA461-m4, and OSA024 strains, among the nopP gene function-deficient strains obtained in the present invention as rhizobia that avoid incompatibility with Rj2 / GmNNL1 soybean, have extremely high nodule occupancy abilities under competitive inoculation conditions with multiple other rhizobia. Furthermore, FY2-m1, GMA461-m4, and OSA024 strains occupied more than 90% of the N of Rj2 / GmNNL1 soybean. ２ O emissions were significantly reduced compared to FY2-m1, GMA461-m4, and rj2 / Gmnnl1 soybeans with low OSA024 strain occupancy. ２ From the viewpoint of O reduction, it can be said that the GMA461-m4, FY2-m1, and OSA024 strains are superior.

[0151] Example 7 The nopP gene function-deficient strains GMA461-m4, FY2-m1, and OSA024 were each suspended in sterilized water to a total cell count of 1.0 x 10 ９ A microbial material adjusted to 100 cells / mL was prepared. Four seeds of chlorine-fumigated Rj2 / GmNNL1 soybeans were sown in Wagner pots containing 3 L of NARO field soil, and 1 mL of the microbial material prepared above was administered per seed. Eight pots were prepared per rhizobia strain. Cultivation was carried out in a greenhouse (25°C) under conditions of 16 hours of light and 8 hours of darkness. Four days after sowing, the soybeans were thinned out, leaving only two Rj2 / GmNNL1 soybeans with good germination in each Wagner pot. Cultivation continued for 38 days. Tap water was periodically supplied to the Wagner pots during cultivation.

[0152] After 38 days of cultivation, six soybean plants were harvested from three pots for each rhizobia strain, the above-ground parts were excised, the roots were washed with tap water, and the number of mature nodules formed on the roots for each rhizobia strain was counted. The mature nodules were then collected and immersed in a 0.5% sodium hypochlorite solution for 3 minutes to sterilize the nodule surface. Each mature nodule was then crushed, and DNA was extracted from each mature nodule. The DNA was subjected to PCR analysis to detect the Bradyrhizobium ottawaense-type nosZ gene, and the proportion of mature nodules occupied by the inoculated rhizobia in the mature nodules (nodule occupancy rate) was quantified.

[0153] The soybean plants in the remaining five pots for each rhizobia were excised to promote rhizosphere senescence. One day after excision of the soybean plants, the N released from each Wagner pot was measured every 2-3 days. ２ O emission amount (N ２ The above N ２ The amount of O released was measured using a mid-infrared laser gas monitor.

[0154] Comparative Example 6 The same procedure as in Example 7 was carried out, except that sterilized distilled water was administered to the Wagner pot instead of the microbial material.

[0155] Figure 13(A) shows the nodule occupancy rates of the GMA461-m4, FY2-m1, and OSA024 strains of Example 7. As shown in Figure 13(A), FY2-m1 and GMA461-m4 showed nodule occupancy rates of over 80%, and OSA024 showed a nodule occupancy rate of just under 60%. Figure 13(B) shows the N over time from Wagner pots to which the microbial material containing the GMA461-m4, FY2-m1, or OSA024 strain of Example 7 or the sterilized distilled water of Comparative Example 6 was administered. ２ O release amount (μgNm －２ h －１ ) ± standard error. The asterisk in Figure 13(B) indicates data for which a significant difference was detected by Dunnett's test using Mock as a control. In Figure 13, "Mock" refers to the group of Wagner pots to which the sterilized distilled water was administered. N from Wagner pots to which the microbial material containing the GMA461-m4, FY2-m1, or OSA024 strain was administered ２ The amount of O released was measured using a Wagner pot dosed with sterile distilled water. ２ The N2O3 release from the Wagner pots to which the microbial materials containing the FY2-m1 or GMA461-m4 strains were administered tended to be lower than the O2 release, and in particular, the nodule occupancy rate was over 80% in Figure 13(A). ２ 13(C) shows the cumulative N released from Wagner pots dosed with the microbial materials containing the GMA461-m4, FY2-m1, or OSA024 strains of Example 7, or the sterilized distilled water of Comparative Example 6. ２ O release amount (kgNha －１"ha" (hectare) is 10,000m ２ 13(C) shows the mean ± standard deviation of the cumulative N in the GMA461-m4, FY2-m1, and OSA024 strains of Example 7. In addition, in FIG. 13(C), data with a p value of less than 0.05 as a result of Dunnett's test using mock as a control are marked with one asterisk, and data with a p value of less than 0.01 are marked with two asterisks. As shown in FIG. 13(C), the cumulative N in the GMA461-m4, FY2-m1, and OSA024 strains of Example 7 ２ The average value of the cumulative N O release amount in the sterilized distilled water of Comparative Example 6 is ２ The mean values ​​of O release were significantly lower than those of the mean values ​​of O release.

[0156] (1) Name of depository institution: National Institute of Technology and Evaluation Patent Microorganism Deposit Center (2) Contact: 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818 (3) Accession number: NITE BP-04046, NITE BP-04047, NITE BP-04048, NITE BP-03361 (4) Identification: FY2-m1 (NITE BP-04046), GMA461-m4 (NITE BP-04047), OSA024 (NITE BP-04048), SG09 (NITE BP-03361) (5) Date of deposit: December 14, 2023 (NITE BP-04046, NITE BP-04047, NITE BP-04048) (6) Transfer date: November 8, 2021 (NITE BP-03361)

[0157] [Rule 26, amended 29.01.2025]

Claims

1. A rhizobia in which the function of at least one effector gene that induces symbiotic incompatibility with legumes is deleted or reduced.

2. The root nodule bacteria of claim 1, which is a Bradyrhizobium bacterium.

3. The root nodule bacterium according to claim 2, which is Bradyrhizobium ottawaense.

4. The rhizobia according to any one of claims 1 to 3, wherein the loss of gene function is due to a transposon insertion in the NopP gene or a mutation in the amino acid sequence of NopP.

5. The rhizobia described in claim 4, wherein the mutation in the amino acid sequence of NopP is due to the substitution of the amino acid residue corresponding to position 60 of the amino acid sequence of SEQ ID NO: 2 with leucine and the amino acid residue corresponding to position 271 with phenylalanine.

6. The rhizobia according to any one of claims 1 to 3, wherein NopP is a protein in which the amino acid residue corresponding to position 60 in SEQ ID NO: 2 is substituted with leucine and the amino acid residue corresponding to position 271 is substituted with phenylalanine in the FY2-m1 strain (accession number NITE BP-04046), the GMA461-m4 strain (accession number NITE BP-04047), or the OSA024 strain (accession number NITE BP-04048), or the Bradyrhizobium ottawaense SG09 strain (accession number NITE BP-03361).

7. A method for infecting a legume plant with rhizobia, the method comprising inoculating a legume plant expressing two or more symbiotic incompatibility genes with rhizobia in which the function of at least one effector gene that induces symbiotic incompatibility with the legume plant is deleted or reduced.

8. The method of claim 7, wherein the root nodule bacteria is a Bradyrhizobium bacterium.

9. The method of claim 8, wherein the root nodule bacteria is Bradyrhizobium ottawaense.

10. The method according to any one of claims 7 to 9, wherein the loss of gene function is due to a transposon insertion in the NopP gene or due to a mutation in the amino acid sequence of NopP.

11. The method according to claim 10, wherein the mutation in the amino acid sequence of NopP is due to substitution of the amino acid residue corresponding to position 60 of the amino acid sequence of SEQ ID NO: 2 with leucine and the amino acid residue corresponding to position 271 with phenylalanine.

12. The method according to any one of claims 7 to 9, wherein the rhizobia is a strain of FY2-m1 (accession number NITE BP-04046), GMA461-m4 (accession number NITE BP-04047), or OSA024 (accession number NITE BP-04048), or a strain of Bradyrhizobium ottawaense SG09 (accession number NITE BP-03361), in which NopP is a protein in which the amino acid residue corresponding to position 60 in SEQ ID NO: 2 is substituted with leucine and the amino acid residue corresponding to position 271 is substituted with phenylalanine.

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