Microbes and Environments
Online ISSN : 1347-4405
Print ISSN : 1342-6311
ISSN-L : 1342-6311
Regular Paper
Inoculation with Bradyrhizobium ottawaense Effectively Reduces N2O Emissions from Degraded Soybean Nodules in Andosols Dominated by nosZ-lacking Bradyrhizobia
Misa Tsubokura, Shintaro Hara, Tsubasa Ohbayashi, Masanori Takeda, Yuma Sasaki, Haruka Ukita, Miho Fujimori, Kaori Kakizaki, Manabu Itakura, Kiwamu Minamisawa, Hiroko Akiyama
著者情報
キーワード: N2O mitigation, Bradyrhizobium ottawaense, N2O reductase gene (nosZ), soybean nodule, pot experiment
ジャーナル オープンアクセス HTML
電子付録

2026 年 41 巻 3 号 論文ID: ME26022

詳細
PDFをダウンロード (2775K)
メタデータをダウンロード RIS形式

(EndNote、Reference Manager、ProCite、RefWorksとの互換性あり)

BIB TEX形式

(BibDesk、LaTeXとの互換性あり)

テキスト
メタデータのダウンロード方法
発行機関連絡先
Abstract

Nitrous oxide (N2O) is a potent greenhouse gas, and its largest anthropogenic source is agricultural soils. In these soils, N2O emissions are produced from nitrogen fertilization and nitrogen-rich crop residues, such as soybean nodules. Previous studies demonstrated that inoculation with Bradyrhizobium diazoefficiens possessing the N2O reductase gene (nosZ) effectively mitigated N2O emissions from soybean fields at harvest. More recently, Bradyrhizobium ottawaense, which exhibits higher nosZ expression, reduced N2O emissions more effectively than B. diazoefficiens during the degradation of soybean nodules formed under sterile vermiculite conditions. Although the effects of inoculation with B. ottawaense have subsequently been exami­ned under natural soil conditions, it remains unclear whether these effects are maintained across different soil types and soybean cultivars. Therefore, we herein investigated the effects of inoculation with B. ottawaense on its competitiveness and N2O emissions in pot experiments using two field soils (Andosol and Fluvisol) and three Japanese commercial soybean cultivars (‘Hatayutaka’, ‘Yukihomare R’, and ‘Satonohohoemi’). The results demonstrated that inoculation with B. ottawaense significantly increased higher nodule occupancy of the inoculant strain compared with the non-inoculated controls in the two field soils. Moreover, B. ottawaense tended to reduce N2O emissions in Andosol dominated by nosZ-lacking bradyrhizobia, regardless of the soybean cultivar. No significant N2O-reducing effects were observed in Fluvisol dominated by nosZ-possessing bradyrhizobia. Collectively, these results suggest the applicability of inoculation with B. ottawaense to diverse soybean cultivars and its effectiveness in mitigating N2O emissions from soybean fields in which indigenous nosZ-lacking bradyrhizobial populations are dominant, such as Andosols.

Nitrous oxide (N2O) is a potent greenhouse gas with a global warming potential that is 273-fold higher than that of carbon dioxide (Forster et al., 2021). It is also an ozone-depleting substance (WMO, 2022). Approximately 58% of global anthropogenic N2O emissions are due to agricultural activities (Tian et al., 2024), including the application of synthetic fertilizers and manure (Xia et al., 2020; Menegat et al., 2022), and the decomposition of crop residues with a low carbon-to-nitrogen ratio, such as leguminous crops (Huang et al., 2004; Rochette et al., 2004; Akiyama et al., 2020). In agroecosystems, N2O is primarily produced via microbial nitrification and denitrification, which are regulated by soil environmental factors (e.g., nitrogen content, moisture, and pH) (Wrage et al., 2001; Wang et al., 2021a). Consequently, various approaches have been proposed to mitigate N2O emissions, including optimizing nitrogen applications, utilizing enhanced-efficiency fertilizers, improving drainage, and adjusting soil pH through liming (Hassan et al., 2022; Akiyama, 2024). Nevertheless, atmospheric N2O concentrations continue to increase (Tian et al., 2024), highlighting the urgent need to develop innovative mitigation techniques.

Soybean (Glycine max [L.] Merr.) forms root nodules colonized by a group of nitrogen-fixing bacteria in the genus Bradyrhizobium, which supply ammonia fixed from atmospheric nitrogen. In the soybean rhizosphere, N2O emissions occur from degraded nodules during the harvest season (Rochette et al., 2004; Yang and Cai, 2005; Inaba et al., 2012; Akiyama et al., 2016), which are mainly driven by denitrification via microorganisms, including soybean-nodulating bradyrhizobia and soil fungi (Inaba et al., 2012; Moriuchi et al., 2025). Denitrification is a sequential reduction process progressing from NO3–/NO2– to N2O or N2 (Yoon et al., 2019), and N2O is reduced to N2 by N2O reductase encoded by the nosZ gene (Zumft, 1997). Some bradyrhizobial strains lack the nosZ gene (nosZ– bradyrhizobia), and their abundance may be associated with net N2O emissions in the soybean rhizosphere (Uchida and Akiyama, 2013). In Japan, the population of nosZ– bradyrhizobia is abundant in Andosols (volcanic ash soils), which cover approximately half of upland fields (Shiina et al., 2014; Shinjo and Takata, 2021). Accordingly, inoculation with bradyrhizobia possessing the nosZ gene (nosZ+ bradyrhizobia), such as Bradyrhizobium diazoefficiens, has been proposed as an effective option to mitigate N2O emissions in soybean fields. These mitigating effects have been demonstrated in field studies conducted in Japan as well as in South America and France (Itakura et al., 2013; Akiyama et al., 2016; Melissa et al., 2022; Hénault et al., 2022).

In recent years, nosZ+ bradyrhizobia have been identified within Bradyrhizobium ottawaense (Wasai-Hara et al., 2020, 2023). Several strains of B. ottawaense isolated from sorghum roots were shown to exhibit higher N2O-reducing activity than wild-type B. diazoefficiens USDA110T (Wasai-Hara et al., 2023; Itakura and Minamisawa, 2026). Moreover, B. ottawaense was found to be capable of nodulating a wide range of commercial soybean cultivars (Minakata et al., 2023; Win et al., 2023, 2024), and soybean plants inoculated with B. ottawaense showed 43–57% nodule occupancy under field conditions (Hara et al., 2024). Nishida et al. (2025) further demonstrated that incompatibility-bypassing strains of B. ottawaense, combined with soybean lines bred for enhanced host selectivity, achieved >60% nodule occupancy of the inoculant, with significant N2O reduction. Therefore, B. ottawaense is expected to be a highly potent bradyrhizobial species that reduces N2O emissions from soybean fields.

To confirm this potential, it was necessary to investigate the competitiveness and N2O-reducing effects of B. ottawaense across different soil types and commercial soybean cultivars. Therefore, we conducted pot experiments: (i) to identify the strain with superior N2O-reducing activity (Experiment 1); (ii) to examine its inoculation effects in two major soils of Japan (Experiment 2); and (iii) to evaluate its performance in three commercial soybean cultivars developed in Japan (Experiments 3 and 4).

Materials and Methods

Bradyrhizobium strains and culture conditions

The Bradyrhizobium strains used in the present study are listed in Table 1. B. ottawaense strains were isolated from diverse locations and host plants, while the S110 group (mixed strains of B. diazoefficiens isolated from soybean nodules in Hokkaido) was selected to cover a wide range of genomic diversity based on a comparative genomic anal­ysis (data not shown). B. diazoefficiens strain TSU1-4 (previously described as ‘TS1-4’ by Shiina et al., 2014 and Hara et al., 2024), which is phylogenetically close to B. diazoefficiens USDA110T, was isolated from a soybean nodule in Ibaraki. All strains were cultured at 30°C with agitation at 110 rpm in HM broth (Cole and Elkan, 1973) supplemented with 1 g L–1 arabinose and 0.25 g L–1 yeast extract.

Table 1.Bradyrhizobial strains used in this study

Strain name*1 Species Isolation site Host plant nosZ Nodulation*2 Accession No. (genome) Reference
TSU1-4 (TS1-4) Bradyrhizobium diazoefficiens Tsukuba, Ibaraki Soybean + + — Shiina et al., 2014; Hara et al., 2024; Toyoda et al., 2024,
SF21 Bradyrhizobium ottawaense Nihonmatsu, Fukushima Sorghum + + SAMD00634907 Wasai-Hara et al., 2023
SG09 Bradyrhizobium ottawaense Nihonmatsu, Fukushima Sorghum + + SAMD00179679 Wasai-Hara et al., 2020; 2023
SG10 Bradyrhizobium ottawaense Nihonmatsu, Fukushima Sorghum + + SAMD00634909 Wasai-Hara et al., 2020; 2023
SG23 Bradyrhizobium ottawaense Nihonmatsu, Fukushima Sorghum + + SAMD00634900 Wasai-Hara et al., 2020; 2023
SH12 Bradyrhizobium ottawaense Nihonmatsu, Fukushima Sorghum + + SAMD00634901 Wasai-Hara et al., 2023
FA1 Bradyrhizobium ottawaense Numata, Gunma Soybean + + — Itakura et al., 2026
FY1 Bradyrhizobium ottawaense Numata, Gunma Soybean + + — Itakura et al., 2026
FY2 Bradyrhizobium ottawaense Numata, Gunma Soybean + + — Nishida et al., 2025; Itakura et al., 2026
S110: bacterial mixture of 10 strains of B. diazoefficiens
ZK1 Bradyrhizobium diazoefficiens Kamishihoro, Hokkaido Soybean + + SAMD01705412 This study
ZK4 Bradyrhizobium diazoefficiens Kamishihoro, Hokkaido Soybean + + SAMD01705413 This study
ZK7 Bradyrhizobium diazoefficiens Kamishihoro, Hokkaido Soybean + + SAMD01705414 This study
ZK12 Bradyrhizobium diazoefficiens Kamishihoro, Hokkaido Soybean + + SAMD01705415 This study
ZK14 Bradyrhizobium diazoefficiens Kamishihoro, Hokkaido Soybean + + SAMD01705416 This study
ZK17 Bradyrhizobium diazoefficiens Kamishihoro, Hokkaido Soybean + + SAMD01705417 This study
ZK18 Bradyrhizobium diazoefficiens Kamishihoro, Hokkaido Soybean + + SAMD01705418 This study
ZK19 Bradyrhizobium diazoefficiens Kamishihoro, Hokkaido Soybean + + SAMD01705419 This study
ZK23 Bradyrhizobium diazoefficiens Kamishihoro, Hokkaido Soybean + + SAMD01705420 This study
ZK25 Bradyrhizobium diazoefficiens Kamishihoro, Hokkaido Soybean + + SAMD01705421 This study

*1 Sequence identities between strains of Bradyrhizobium diazoefficiens and Bradyrhizobium ottawaense were similar, according to a phylogenetic anal­ysis based on 16S–23S rRNA intergenic transcribed spacer (ITS) region sequences (Fig. S1).

*2 Nodulation ability was evaluated using an inoculation test with soybean.

Pot experimental designs

Four pot experiments (Experiments 1–4) were performed in greenhouses under natural light at the Institute for Agro-Environmental Sciences, National Agriculture and Food Research Organization (NIAES, NARO) in Tsukuba, Ibaraki, Japan (36°0′N, 140°1′E). Two agricultural soils: Andosol and Fluvisol (also called Gray lowland soil in the Soil Classification System in Japan) were collected from the experimental fields at NIAES and sieved through a 4.75-mm mesh. The properties of Andosol and Fluvisol were previously described by Akiyama et al. (2020) and Toyoda et al. (2024), respectively. In Experiments 1 and 2, soils were filled into biodegradable Jiffy pots (approximately 0.3 L; Jiffy International AS), which were then placed in 1/5,000 a Wagner pots filled with vermiculite. In Experiment 3, 3 L of Andosol was directly filled into 1/5,000 a Wagner pots. These experiments used two Japanese commercial soybean cultivars: ‘Hatayutaka’ (medium-maturing) and ‘Yukihomare R’ (early-maturing), which were developed for the central (Kanto) and northern (Hokkaido) regions of Japan, respectively.

Experiment 1:

Experiment 1 consisted of two independent evaluations for analyzing nodule occupancy (Experiment 1-1, Fig. 1A) and measuring N2O flux (Experiment 1-2, Fig. 1B). These evaluations were conducted to compare the inoculation effects of eight B. ottawaense strains (SF21, SG09, SG10, SG23, SH12, FA1, FY1, or FY2) with B. diazoefficiens TSU1-4 in Andosol.

Fig. 1. Effects of bradyrhizobial inoculations on nodule occupancy and N2O emission in Experiment 1. (A) Overview of Experiment 1. Treatments are non-inoculation (None), inoculation with B. diazoefficiens strain (TSU1-4) or B. ottawaense strain (any of SF21, SG09, SG10, SG23, SH12, FA1, FY1, or FY2). (A) Overview of Experiment 1-1 for analyzing nodule occupancy. DAS: days after sowing. (B) Overview of Experiment 1-2 for monitoring N2O emissions. (C) Cumulative N2O emissions from Andosol for 28 days from shoot decapitation. “BD type” and “BW type” indicate “Bradyrhizobium diazoefficiens” and “Bradyrhizobium ottawaense”, respectively. Values are means±SD (n=4 replicate pots). Different characters indicate significant differences (P<0.05); the significance of differences was evaluated using Tukey’s HSD test. Nodule occupancy rates by nosZ+ bradyrhizobia are shown in Fig. S2A.

Experiment 1-1:

Dry soybean seeds (cv. Hatayutaka) were covered with moist vermiculite at room temperature for 1 day for pre-germination, and four seeds were then sown in the biodegradable pots. One milliliter of a bacterial suspension (1×109‍ ‍cells‍ ‍mL–1) containing a strain of B. ottawaense or B. diazoefficiens, or sterile deionized water (as a non-inoculated control) was applied to each seed. Each treatment was performed in four replicates. At 13 days after sowing (DAS), one seedling was left per pot by thinning. At 37 DAS, shoot decapitation was performed. The nodules were counted and weighed per pot. They were then immediately frozen using liquid nitrogen and stored at –80°C until the PCR anal­ysis.

Experiment 1-2:

The pot preparation and bradyrhizobial inoculation were performed as described in Experiment 1-1. The seedlings were thinned at 10 DAS. Shoot decapitation was performed at 41 DAS, and the shoot parts were then dried and weighed. Gas sampling was conducted for 28 days after decapitation in the greenhouses.

Experiment 2:

Experiment 2 was conducted to examine differences in the inoculation effect of B. ottawaense SG09 between two field soils (Fig. 2A). Four dry soybean seeds (cv. Hatayutaka) were sown in biodegradable pots containing Andosol or Fluvisol, and the inoculation with B. ottawaense SG09 was then performed as described above. Each treatment was performed in ten replicates. Two of the four seedlings were thinned per pot at 7 DAS. Shoot decapitation was conducted at 31 DAS, and the shoot parts were then dried and weighed. The nodules in five of the ten pots were counted, weighed, and then stored until the PCR anal­ysis as described in Experiment 1-1. Nodule occupancy rates were investigated by nosZ multiplex PCR and an ITS-RFLP anal­ysis in three of the five pots. Gas sampling of the five other pots was conducted for 20 days in the greenhouses.

Fig. 2. Effects of bradyrhizobial inoculations with two field soils in Experiment 2. (A) Overview of Experiment 2. Treatments are non-inoculation (None) or an inoculation with Bradyrhizobium ottawaense SG09 in Andosol or Fluvisol. (B, C) Nodule occupancy rates of nosZ+ bradyrhizobia in (B) Andosol and (C) Fluvisol. (n=3 replicate pots). The total number of analyzed nodules is shown in parentheses under each treatment label. More detailed nodule occupancy rates are shown in Fig. S3. (D, E) Cumulative N2O emissions from (D) Andosol and (E) Fluvisol for 20 days from shoot decapitation. Values are means±SD (n=5 replicate pots). ‘n.s.’ means no significant difference (P>0.05); the significance of differences was evaluated using the t-test.

Experiment 3:

Experiment 3 was conducted to evaluate the host cultivar-dependent effects of the inoculation with B. ottawaense SG09 (Fig. 3A). The inoculation effect of SG09 was compared with those of B. diazoefficiens TSU1-4 and the S110 group. Four dry soybean seeds (cv. Hatayutaka or cv. Yukihomare R) were sown in 1/5,000 a Wagner pots containing Andosol, and the bradyrhizobial inoculation was then performed as described above. Each treatment was conducted in eight replicates. Two of the four seedlings were thinned at 14 DAS. Shoot decapitation was performed at 46 DAS, and the shoot parts were then dried and weighed. The nodules in four of the eight pots were counted, weighed, and stored until the PCR anal­ysis as described in Experiment 1-1. Nodule occupancy rates were subsequently investigated by nosZ multiplex PCR. Gas sampling of the four other pots was conducted for 45 days in the greenhouses.

Fig. 3. Effects of bradyrhizobial inoculations with two soybean cultivars in Experiment 3. (A) Overview of Experiment 3. Treatments for Experiment 3 were non-inoculation (None), an inoculation with Bradyrhizobium diazoefficiens TSU1-4 or the S110 group, or an inoculation with Bradyrhizobium ottawaense SG09 for ‘Hatayutaka’ or ‘Yukihomare R’. (B, C) Nodule occupancy rates of nosZ+ bradyrhizobia in nodules of (B) ‘Hatayutaka’ and (C) ‘Yukihomare R’. (n=4 replicate pots). (D, E) Cumulative N2O emissions from Andosol with nodules of (D) ‘Hatayutaka’ and (E) ‘Yukihomare R’ for 45 days after shoot decapitation. Values are means±SD (n=4 replicate pots). Different characters indicate significant differences (P<0.05); the significance of differences was evaluated using Tukey’s HSD test.

Further details of methods and a description of Experiment 4 are provided in the Supplementary Materials.

Analysis of nodule occupancy by nosZ multiplex PCR and the ITS-RFLP anal­ysis

The nodule occupancy rates of inoculated B. diazoefficiens and B. ottawaense carrying the nosZ gene were assessed by species-specific multiplex PCR, as previously described (Hara et al., 2024). Briefly, DNA templates were prepared from crushed nodules in 96-well plates using crude extraction and a proteinase K treatment. Multiplex PCR was conducted with nosZ-specific primer sets (nosZ_BD-F/R for B. diazoefficiens and nosZ_BW-F/R for B. ottawaense; Table S1). In the RFLP anal­ysis of the 16S-23S rRNA gene internal transcribed region (the ITS-RFLP anal­ysis), PCR was conducted using ITS primers (BraITS-F and BraITS-R; Table S1) to simultaneously detect Bradyrhizobium japonicum and Bradyrhizobium elkanii species, as previously described (Saeki et al., 2006; Itakura et al., 2026), and the PCR products were subjected to RFLP typing with the restriction enzyme MspI (TaKaRa Bio).

N2O flux measurement

N2O fluxes were measured every 3–4 days after decapitation. Gas sampling was always conducted at 9–10 a.m. to minimize diurnal fluctuations (Alves et al., 2012). Headspace gases (30 mL) from each pot were collected using a 50-mL syringe at 0, 20, and 40 min after sealing with acrylic lids equipped with syringe septa. Samples were immediately injected into evacuated 15-mL vials. N2O concentrations were analyzed using a gas chromatograph (GC-2014; Shimadzu; Sudo and Yamamoto, 2017), in combination with a CH4-doped 63Ni electron capture detector at 340°C using a headspace auto-sampler (AOC-5000; Shimadzu; Sudo and Yamamoto, 2017). Nitrogen was used as the carrier gas. Fluxes were calculated from the linear change in N2O concentrations across the three time points (Hou et al., 2016), while cumulative N2O emissions were calculated using the trapezoidal integration method.

Statistical anal­ysis

All statistical anal­yses were conducted using R software (version 4.3.1) (R Core Team, 2023). P values <0.05 were considered to be significant. The statistical methods used are indicated in each figure legend, and a detailed anal­ysis method is provided in the Supplementary Materials.

Genomic sequence accession numbers

The 16S rRNA gene sequences of the S110 group were deposited in the DDBJ Sequence Read Archive under accession numbers SAMD01705412–SAMD01705421 (Table 1).

Results

Experiment 1: N2O-reducing potential of eight B. ottawaense strains

In Experiment 1-1, the inoculations with each of the eight B. ottawaense strains and with B. diazoefficiens showed the significantly higher occupancy of nosZ+ bradyrhizobial inoculants, respectively, than the non-inoculated control (Fig. S2A, Table 2). On the other hand, the nosZ– bradyrhizobial population was dominant in the non-inoculated control. In Experiment 1-2, cumulative N2O emissions were significantly lower with the inoculations with each B. ottawaense strain (71.5–87.9% reductions) than with the non-inoculation (Fig. 1C and S2B, Table 2). No significant differences were observed in nodule occupancy or N2O reduction rates among the B. ottawaense strains and B. diazoefficiens strain. There were also no significant differences in the number and fresh weight of nodules or shoot dry weight among the treatments (Fig. S2C, D, and E). Soybean plants reached the reproductive stage at the time of decapitation regardless of the bradyrhizobial inoculation in both experiments. Collectively, these results demonstrate that the eight B. ottawaense strains used in this study exhibited similar capabilities for nodulation and N2O reduction.

Table 2.Nodule occupancy rate by nosZ-possessing bradyrhizobia and cumulative N2O emissions in each experiment.

Experiment No. Treatment Soil type Soybean cultivar Nodule occupancy by nosZ PCR (%)*1 Cumulative N2O emission
(g-N ha–1)*1		 N2O reduction rate (%)*2
Inoculated strain nosZ type of bradyrhizobia BD type (%) BW type (%)
Experiment 1 None (control) — — Andosol Hatayutaka 1.6±1.9 b 4.0±6.1 b 90.0±51.0 a —
TSU1-4 nosZ+ BD-type Andosol Hatayutaka 92.8±4.9 a 0.0±0.0 b 16.9±7.8 b 81.3
SF21 nosZ+ BW-type Andosol Hatayutaka 0.0±0.0 b 82.3±12.5 a 14.1±15.6 b 84.3
SG09 nosZ+ BW-type Andosol Hatayutaka 0.8±1.6 b 80.6±8.7 a 18.3±12.2 b 79.7
SG10 nosZ+ BW-type Andosol Hatayutaka 0.0±0.0 b 81.5±8.5 a 16.7±8.9 b 81.5
SG23 nosZ+ BW-type Andosol Hatayutaka 0.0±0.0 b 90.0±9.5 a 10.9±2.2 b 87.9
SH12 nosZ+ BW-type Andosol Hatayutaka 0.0±0.0 b 69.4±12.5 a 25.6±13.7 b 71.5
FA1 nosZ+ BW-type Andosol Hatayutaka 0.8±1.6 b 70.2±14.7 a 18.4±13.1 b 79.6
FY1 nosZ+ BW-type Andosol Hatayutaka 0.0±0.0 b 80.6±13.7 a 20.1±17.3 b 77.6
FY2 nosZ+ BW-type Andosol Hatayutaka 1.6±1.9 b 66.1±17.6 a 21.8±13.0 b 75.8
Experiment 2# None (control) — — Andosol Hatayutaka 3.3±3.8 n.s. 0.0±0.0 b 218.9±157.8 a —
SG09 nosZ+ BW-type Andosol Hatayutaka 1.7±1.4 n.s. 63.3±11.8 a 90.6±42.9 a 58.6
None (control) — — Fluvisol Hatayutaka 59.2±3.8 a 3.3±1.4 b 64.3±36.1 a —
SG09 nosZ+ BW-type Fluvisol Hatayutaka 5.1±3.7 b 83.0±6.3 a 58.3±15.5 a 9.4
Experiment 3 None (control) — — Andosol Hatayutaka 8.3±8.8 c 5.8±5.7 b 347.4±59.3 a —
TSU1-4 nosZ+ BD-type Andosol Hatayutaka 62.5±19.9 a 0.0±0.0 b 61.3±8.7 b 82.4
S110 nosZ+ BD-type Andosol Hatayutaka 56.7±7.2 b 4.2±1.7 b 96.2±80.2 b 72.3
SG09 nosZ+ BW-type Andosol Hatayutaka 1.7±3.4 c 59.2±14.2 a 108.6±40.9 b 68.7
None (control) — — Andosol Yukihomare R 9.2±6.9 c 0.0±0.0 b 557.0±262.3 a —
TSU1-4 nosZ+ BD-type Andosol Yukihomare R 76.7±20.2 a 0.0±0.0 b 112.5±47.0 b 79.8
S110 nosZ+ BD-type Andosol Yukihomare R 45.0±12.9 b 0.0±0.0 b 264.0±60.1 b 52.6
SG09 nosZ+ BW-type Andosol Yukihomare R 10.0±9.8 c 28.3±25.7 a 173.4±47.7 b 68.9

*1 Values are means±SD (nodule occupancy rate: n=4 or 3#, cumulative N2O emission: n=4 or 5#). Nodule occupancy rates by Bradyrhizobium diazoefficiens (BD type) or Bradyrhizobium ottawaense (BW type) and cumulative N2O emissions were statistically evaluated using Tukey’s HSD test (Experiments 1 and 3) or the t-test (Experiment 2). Different letters indicate significant differences (P<0.05).

*2 N2O reduction rate (%)=(1–(total cumulative N2O emissions in the inoculated treatment)/(total cumulative N2O emissions in the non-inoculated treatment))×100

Total cumulative N2O emissions represent the sum across all replicates in each treatment.

Of these strains, various features of B. ottawaense SG09 have been extensively exami­ned, such as nodule formation, N2O-reducing activity, and the mechanisms underlying attachment to plant roots (Wasai-Hara et al., 2023; Win et al., 2023, 2024; Hara et al., 2024; Toyoda et al., 2024; Nishida et al., 2025; Takeguchi et al., 2025; Itakura and Minamisawa, 2026). Therefore, strain SG09 was selected for subsequent experiments.

Experiment 2: Effects of the B. ottawaense inoculation on N2O emissions from degraded soybean nodules in Andosol and Fluvisol

The nodule occupancy rates of B. ottawaense in the SG09-inoculated treatment were 63.3 and 83.0% in Andosol and Fluvisol, respectively (Fig. 2B and C, Table 2). In contrast, almost no occupancy of B. ottawaense was observed in the non-inoculated control in either soil, and the predominant bradyrhizobial species were B. elkanii (nosZ–) (71.7%) in Andosol and B. diazoefficiens (59.6%) in Fluvisol (Fig. 2C and S3, Table 2).

Maximum N2O fluxes in Andosol were 47.6±25.3 and 148.1±131.7 μg N2O-N m–2 h–1 from the non- and SG09-inoculated pots, respectively, at 10 days after decapitation (Fig. S4A). Cumulative N2O emissions were 58.6% lower with the SG09 inoculation than with the non-inoculation (Table 2), although this difference was not significant due to large variations in the non-inoculated treatment (Fig. 2D). N2O fluxes were markedly lower in Fluvisol than in Andosol regardless of the bradyrhizobial inoculation, and no significant difference was observed in cumulative N2O emissions between the non- and SG09-inoculated treatments (Fig. 2E, Table 2). A two-way ANOVA of cumulative N2O emissions revealed a significant effect of soil type (P=0.024), but not of bradyrhizobial inoculation (P=0.093). No interaction was detected between the two factors (P=0.124).

Shoot dry weight and nitrogen content in both soils were similar with the SG09 inoculation and non-inoculation (Fig. S4B and C). However, in Fluvisol, SG09-inoculated soybean plants formed significantly more nodules with a greater fresh weight than non-inoculated plants, whereas no significant differences were observed in Andosol (Fig. S4D and E). Furthermore, the developmental stages of plants were the same in both soils (vegetative stage) at the time of decapitation. These results indicate that the SG09-inoculated treatment had no effect on plant growth.

Experiment 3: Effects of the B. ottawaense inoculation on N2O emissions in different soybean cultivars

The nodule occupancy rates of B. ottawaense were 59.2 and 28.3% in ‘Hatayutaka’ and ‘Yukihomare R’, respectively, under the SG09-inoculated treatment (Fig. 3B and C, Table 2). In contrast, the occupancy of B. ottawaense was negligible in both cultivars in the non-, TSU1-4-, and S110-inoculated treatments (0.0–5.8%). The TSU1-4- and S110-inoculated treatments increased the occupancy of B. diazoefficiens in both cultivars significantly more than the respective non-inoculated controls (45.0–76.7%).

Cumulative N2O emissions in non-inoculated treatments differed between soybean cultivars: soils cultivated with ‘Yukihomare R’ showed 1.6-fold higher emissions than those cultivated with ‘Hatayutaka’ (Fig. 3D and E, Table 2). Despite this difference, the SG09-, TSU1-4-, and S110-inoculated treatments reduced N2O emissions from each soil cultivated with ‘Hatayutaka’ and ‘Yukihomare R’ significantly more than their respective non-inoculated controls (Fig. 3D, 3E, S5A, and S5B). The reduction rates of these nosZ+ bradyrhizobial-inoculated treatments were similar between the two cultivars (Table 2). The number of nodules was higher in ‘Yukihomare R’ than in ‘Hatayutaka’, and the TSU1-4-inoculated treatment tended to promote nodule formation more than the other treatments in both cultivars (Fig. S6A and B). A two-way ANOVA showed the significant effects of soybean cultivar (P<0.0001) and bradyrhizobial inoculation (P=0.0127) on the number of nodules, with no interaction (P=0.539). The developmental stages of the two cultivars at decapitation differed regardless of the inoculants: ‘Hatayutaka’ was at the vegetative stage, while ‘Yukihomare R’ had reached the reproductive stage. However, shoot dry weight was not significantly affected by the bradyrhizobial inoculation (Fig. S6C and D).

Discussion

All experiments in the present study showed that the inoculation with B. ottawaense resulted in significantly higher nodule occupancy than with the non-inoculated controls (Table 2). However, the N2O-reducing effects of B. ottawaense may vary depending on soil type. Strain SG09 tended to reduce N2O emissions in Andosol, but not in Fluvisol (Fig. 2D and E). This variation may be explained by differences in the abundance of indigenous nosZ+ bradyrhizobia between the two soils. In agricultural fields of Japan, nosZ– bradyrhizobia dominate Andosols with a porous structure and lower bulk density, whereas nosZ+ bradyrhizobia dominate alluvial soils with a dense structure, such as Fluvisols (Shiina et al., 2014; Akiyama et al., 2020; Takata et al., 2021). Since nosZ+ bradyrhizobia performing N2O respiration—most of which belong to B. diazoefficiens commonly found in agricultural soils—are frequently detected under anaerobic conditions that develop in fine-textured soils (Saeki et al., 2013), Fluvisols are likely to have an advantage in their survival. In the present study, since N2O emissions were minimal in Fluvisols where indigenous nosZ+ bradyrhizobia were already abundant, the N2O-reducing effects of SG09 appeared to be limited. Therefore, inoculation with B. ottawaense may be effective in reducing N2O emissions from soybean fields with a low abundance of indigenous nosZ+ bradyrhizobia.

In addition, cumulative N2O emissions were higher in the treatments using ‘Yukihomare R’ than in those using ‘Hatayutaka’ (Fig. 3D and E, Table 2), which may be attributed to differences in nodule number. Nodule number is a key factor affecting N2O production (Kim et al., 2005) and markedly increases after the reproductive stage (Weil and Ohlrogge, 1975; Takahashi et al., 2005; Arachchige et al., 2020). ‘Yukihomare R’ reached the reproductive stage earlier and formed more nodules than ‘Hatayutaka’ (Fig. S6A and B), which may have contributed to higher N2O production. However, the N2O-reducing effects of SG09 were consistent regardless of differences in N2O emission levels. Therefore, inoculation with B. ottawaense has the potential to achieve a stable N2O reduction across diverse soybean cultivars.

In Experiment 4, the supplemental experiment using ‘Hatayutaka’ and ‘Satonohohoemi’ (Fig. S7 and S8), N2O-reducing effects were less pronounced than in Experiment 3, despite the high occupancy of nosZ+ bradyrhizobia (Table S2). N2O emission levels in the non-inoculated controls of Experiment 4 were also markedly lower than in the other experiments, regardless of the cultivar (Fig. S5C and D). Only Experiment 4 was conducted during winter and had the lowest solar radiation (Table S3). The low light availability during this period may have restricted soybean development, including nodule formation (Wang et al., 2021b), resulting in fewer nodules in Experiment 4 (Fig. S6A, S6B, S8A, and S8B). Consequently, the N2O-reducing effects of nosZ+ bradyrhizobia may not be detectable under extremely low N2O emission levels due to restricted nodule development.

B. ottawaense SG09 possesses two transcription start sites within the nosR transcriptional organization under N2O-respiring conditions (Wasai-Hara et al., 2023). In addition, this strain exhibits a markedly higher maximum N2O reduction rate than B. diazoefficiens USDA110T, which possesses a single transcription start site, thereby contributing to superior N2O-reducing activity observed under both pure liquid culture and sterile soil conditions (Itakura and Minamisawa, 2026). However, the present study using natural soils showed no significant difference in N2O-reducing effects between B. ottawaense and B. diazoefficiens (Fig. 1C, 3D, 3E, S7D, and S7E). A similar discrepancy was reported in previous studies, in which the N2O-reducing effects of nosZ-carrying bacteria were often less effective under soil conditions than under pure culture conditions (Itakura et al., 2013; Usyskin-Tonne et al., 2019). Although the mechanisms underlying this discrepancy remain unclear, it may be attributed to differences between the free-living state in liquid cultures and the bacteroid state within soybean nodules. Further studies are needed to elucidate and overcome the challenges associated with scaling up from pure culture systems to soil environments.

The N2O-reducing effects of nosZ+ bradyrhizobia may be affected by soil moisture. Denitrification-derived N2O production generally increases with the water-filled pore space (WFPS) of soil, particularly between 50–80% (Davidson, 1993). In the present pot study and previous field studies, inoculation with nosZ+ bradyrhizobia mitigated N2O production under the relatively stable WFPS conditions typically associated with denitrification-derived N2O production (WFPS: >40–50%, Fig. S9; Itakura et al., 2013; Akiyama et al., 2016). On the other hand, a recent field study reported no significant N2O reduction following inoculation with B. ottawaense under field conditions with large fluctuations in WFPS (WFPS: 26–71%, Toyoda et al., 2024). Therefore, the effects of soil moisture on bradyrhizobial N2O reduction warrant further study.

In conclusion, inoculation with B. ottawaense significantly increased nodule occupancy and tended to reduce N2O emissions in multiple commercial soybean cultivars under pot-scale soil conditions. Moreover, inoculation with B. ottawaense may be more effective in reducing N2O emissions from Andosol, in which nosZ– bradyrhizobia are dominant, than from Fluvisol, in which nosZ+ bradyrhizobia are dominant. On the other hand, in the present study, the N2O-reducing effects of B. ottawaense were similar to those of B. diazoefficiens, which is in contrast to the findings of a previous laboratory-scale study (Wasai-Hara et al., 2023). To improve the reliability of bradyrhizobial N2O mitigation in agricultural fields, further research is needed to assess the N2O-reducing potential of B. ottawaense under various soil conditions that reflect field environments.

Acknowledgements

The authors thank Yeonghoo Kim for his help with sampling and analyzing the gas, and Mariko Nakagawa and Hikaru Uno for their help with preparing the experimental tools. We are also grateful to the technical support staff of NARO for their support in conducting the experiments.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Sources of Funding

The present study was supported by the Moonshot project JPNP18016, commissioned by New Energy and Industrial Technology Development Organization (NEDO).

Citation

Tsubokura, M., Hara, S., Ohbayashi, T., Takeda, M., Sasaki, Y., Ukita, H., et al. (2026) Inoculation with Bradyrhizobium ottawaense Effectively Reduces N2O Emissions from Degraded Soybean Nodules in Andosols Dominated by nosZ-lacking Bradyrhizobia. Microbes Environ 41: ME26022.

https://doi.org/10.1264/jsme2.ME26022

References
 
© 2026 by Japanese Society of Microbial Ecology / Japanese Society of Soil Microbiology / Taiwan Society of Microbial Ecology / Japanese Society of Plant Microbe Interactions / Japanese Society for Extremophiles.

This article is licensed under a Creative Commons [Attribution 4.0 International] license.
https://creativecommons.org/licenses/by/4.0/
feedback
 Top