佐伯雄一　 宮崎大学農学部

Yuichi Saeki,

Faculty of Agriculture, University of Miyazaki

ダイズ根粒菌の群集生態とN₂Oガス削減

Community Ecology of Soybean Rhizobia and Mitigation of N₂O Emissions

N₂

共生窒素固定

Symbiotic nitrogen fixation

N₂

Study of soybean rhizobia for increase of soybean yield

N₂ + 8H⁺ + 8e^- + 16ATP → 2NH₃ + H₂ + 16 ADP + 16Pi

高窒素固定根粒菌

High N fixing rhizobia

低窒素固定根粒菌

Low N fixing rhizobia

Super Goal

Utilization of rhizobia that have the high activity of symbiotic nitrogen fixation, for increase of soybean yield

High yield

Low yield

窒素固定能の高い有用根粒菌（Bradyrhizbium diazoefficiens）の接種効果とダイズ根粒

Inoculation effects of the highly nitrogen-fixing Bradyrhizobium diazoefficiens and soybean nodules

N₂

N₂

Hokkaido

Akita

Fukushima

Yamanashi

Shizuoka

Kyoto

Kochi

Miyazaki

Ishigaki

Miyako

Okinawa

Tokunoshima

Fukuoka

Soil sampling sites in Japan

それぞれの土壌にダイズを栽培したときに感染する根粒菌の割合を遺伝子型別に色分けしてみる

​

Visualize the proportions of rhizobial genotypes infecting soybean when cultivated in each soil, using color coding for each genotype.

Scheme of isolation of rhizobia from soils and diversity analysis�土壌からの根粒菌分離と多様性解析の概要

sterilized seed

Sample soil 3g

Vermiculite

Culture pot with N-free culture solution

ポット栽培　Pot cultivation

根粒表皮滅菌

Surface sterilization

of nodules

懸濁液の塗布

Homogenization and plating on medium

培養

Incubation

ダイズ根粒菌の分離 Isolation of soybean rhizobia

電気泳動　Electrophoresis

参照株　Reference Strains

B. japonicum USDA 4, 6^T, 38, 115, 123, 124, 135

B. diazoefficiens USDA110^T

B. elkanii USDA 46, 76^T, 94

距離解析Distance analysis

系統樹Dendrogram

Sampling of Soils in Japan

Detection of DNA sequence variance by PCR-RFLP analysis

​

Restriction Fragment Length Polymorphisms of PCR amplicon of genomic sequences

根粒

Nodule

Bj123

Bd110

Bj6

Be76

感染根粒菌群集構造解析を基にした日本の土着ダイズ根粒菌の分布 (Saeki et al. 2013)

Distribution of indigenous soybean-nodulating rhizobia in Japan based on analyses of the community structure of infecting rhizobia

Northern region ：B. japonicum USDA123 line

Central region ：B. diazoefficiens

Western region ：B. japonicum USDA6 line

Southern region：B. elkanii

N₂O

NO

N₂O

Soybean fields cause N₂O emission

Emission of nitric oxide and nitrous oxide

from soybean fields with differing nodulation capacities.

Fig. Changes in nitric oxide, nitrous oxide, and precipitation.

https://www.nies.go.jp/whatsnew/20201005/20201005.html

温暖化係数

Global Warming Potential (GWP)

CO₂ : 1

CH₄ : 30

N₂O : 300

人為的活動による

世界のN₂O排出量

Global N₂O emissions from human activities （2020）

​

CO₂: 3.1✕10¹⁶g/y

​

N₂O: 7.3✕10¹²g/y

農業は、人為的N₂Oガスの発生源

Agriculture is an anthropogenic source of N₂O emission.

The global N₂O balance

Anthropogenic sources

Natural sources

(Source-specific N₂O fluxes)

Anthropogenic emissions

Natural emissions

Agriculture

Natural soil

Ocean

Changes in atmospheric abundance

Atmospheric chemical loss

グライ土や灰色低地土のような水田転換畑に、Bradyrhizobium diazoefficiens（高窒素固定およびN₂O還元活性を有する）に属する分離株が優占化している

Isolates belonging to B. diazoefficiens (Nigh N fixation and N₂O reduction activity) are dominant in paddy-upland rotation fields such as gley soils and gray lowland soils.

日本南北および普通畑と転換畑によるダイズ根粒菌群集構造

N₂O還元活性

N₂O reduction activity

　　　N₂O　→　 N₂

Soybean rhizobial community structure across Japan (north–south) and field types (upland vs. converted paddy fields)

ダイズ根粒菌の中に、N₂O還元能を有する菌が存在する

Some soybean rhizobia possess N₂O-reducing ability.

NO₃^- 　　　　　　　　NO₂^- 　　　　　　　　　NO 　　　　　　　　N₂O 　　　　　　　　N₂

脱窒（硝酸呼吸）Denitrification (nitrate respiration)

napA　　　　　 nirK　　　　　 norB 　　　　　　 nosZ 　

ダイズ根粒菌(B. diazoefficiens USDA110株)のゲノムマップと脱窒遺伝子座

Genome map and denitrification gene locus

Rhizobium

硝酸呼吸を行う４種の酵素をコードする遺伝子がゲノム上に存在する

これらの遺伝子の有無はダイズ根粒菌種により異なる

Genes encoding four types of enzymes involved in nitrate respiration are present in the genome.

These genes are differentially present among soybean rhizobial species.

100bp

ladder

500bp

B. elkanii

B. japonicum

B. diazoefficiens

脱窒活性なし

No denitrification activity

N₂Oまでの不完全脱窒

Incomplete denitrification

N₂まで完全脱窒

Complete denitrification

遺伝子特異的プライマーによる脱窒遺伝子の検出

Detection of denitrification genes by PCR with specific primers

ゲノムマップと脱窒遺伝子座

Genome map and denitrification gene locus

ダイズ根粒菌種や株による脱窒活性の違い

Difference of denitrification ability of bradyrhizobial strains

nosZ+

Bradyrhizobium diazoefficiens

水田転換畑（還元的環境）の土壌で優占

Dominant in paddy field

普通畑（酸化的環境）の土壌で優占

Dominant in upland field

Bradyrhizobium nosZ+ isolates

根粒菌株ごとのN₂O還元活性の測定

N₂O reduction activity of different rhizobial strains.

nosZ+: 水田転換畑で優占

Dominant in paddy field

nosZ-: 普通畑で優占

Dominant in upland field

N₂O reduction activity

ECD-GCによるN₂O分析

N₂O analysis by ECD-GC

nosZ+

nosZ-

N₂O area

Paddy Rice

Soybean

Paddy-Upland Rotation

地球温暖化抑制を可能にするダイズ栽培体系

Climate change–mitigating soybean production systems