LATITUDINAL CHARACTERISTIC NODULE COMPOSITION OF Bradyrhizobium spp. AFFECTED BY THEIR TEMPERATURE-DEPENDENT PROLIFERATION IN SOIL AND INFECTION

概要

（Format No. 13）

SUMMARY OF DOCTORAL THESIS
Name: Md Hafizur Rahman Hafiz
Title:

LATITUDINAL
CHARACTERISTIC
NODULE
COMPOSITION
OF
spp. AFFECTED BY THEIR TEMPERATURE-DEPENDENT
PROLIFERATION IN SOIL AND INFECTION

Bradyrhizobium

(ダイズ根粒菌の温度依存的な土壌中の生息と感染に影響を受ける緯度特異的な根粒内組成)

Soybean (Glycine max [L] Merr.) belongs to the Leguminosae family, and is an
important grain legume. Several soybean-nodulating bacteria, which can fix atmospheric
nitrogen symbiotically in the nodules of the host plant, have been reported worldwide.
Effective nitrogen fixation mainly depends on the potential of the rhizobial strain and
their competition for proliferation and infection among indigenous rhizobia in a soil.
Because effective nitrogen fixation depends on the potential of an individual rhizobial
strain, competition for proliferation and infection among the rhizobial strains in a soil is
crucial subject. In addition, their behaviours need to be considered based on
environmental conditions.

Bradyrhizobium japonicum and B. elkanii are the major soybean nodulating
rhizobia and it has been reported that B. japonicum and B. elkanii dominate in nodules of
soybean cultivated in latitudinally northern and southern fields, respectively. Previous
reports suggest that temperature-dependent infection and proliferation in a soil
determine their nodule compositions. But it has not been elucidated which factor is more
responsible for the latitudinally characteristic nodule composition of the bradyrhizobia.
To examine the contribution of two factors in fields under the local climate
conditions, we selected three study locations, Fukagawa with temperate continental
climate, and Matsue and Miyazaki with humid sub-tropical climate in Japan. Each soil
sample was transported to the other study locations, and soybean cv. Orihime (non-Rj)
was pot-cultivated using three soils at three study locations for successive two years.
Three healthy soybean plants per pot were cultivated in triplicate for ca. 2–3 months
without fertilization, and fresh plant weight and number of nodules were measured at
harvest. Ten nodules were randomly selected from one plant for each replication, and
rhizobial strains were isolated and phylogenetically characterized based on their partial
16S rRNA and 16S–23S rRNA ITS gene sequences, and the nodule composition of
Bradyrhizobium spp. was determined.
The fresh plant weight and the nodule numbers were not significantly different
among soils in both years in all locations with a few exceptions. Overall, the fresh plant
weight increased from northern to southern study locations, whereas the nodule number
showed the opposite tendency. Reduction in nodules at higher temperature might be due
to strain-specific properties.
In this study, two Bradyrhizobium japonicum (Bj11 and BjS10J) and one B. elkanii
(BeL7) were isolated, and Bj11 and BjS10J were phylogenetically sub-grouped into two
(Bj11-1-2) and four clusters (BjS10J-1-4) based on their ITS sequence, respectively.
Bj11-1 was characterized as slow grower and isolated primarily in the Fukagawa soil,
while Bj11-2 was fast grower and isolated in the Fukagawa and Matsue soils. Regarding
BjS10J, BjS10J-1 and BjS10J-3 were originated from the Matsue soil, while BjS10J-2 and
BjS10J-4 were primarily isolated from the Miyazaki soil. B. elkanii L7 was ubiquitous in
all soils.

In the Fukagawa soil, Bj11-1 dominated (87%) in the Fukagawa location, and the
dominancy was not changed in the Matsue (80%) and Miyazaki (83%) locations. In the
Matsue soil, the composition was similar in the Matsue and Miyazaki locations, in which
BeL7 was dominated (70–73%) and Bj11-2 was minor (17-20%). While in the Fukagawa
location, BeL7 decreased to 53% along with the increase of Bj11-1 (17%) and BjS10J-3
(13%). In the Miyazaki soil, BeL7 dominated at 77%, and BeL7 decreased to 13% and 33%
in the Fukagawa and Matsue locations, respectively, while BjS10J-2 (53-73%) and
BjS10J-4 (13%) increased.
These results suggested that the B. japonicum strain preferably proliferated in the
Fukagawa location, leading to its nodule dominancy because the nodule composition was
not changed in the Matsue and Miyazaki locations. While in the Miyazaki location, B.
elkanii dominated in the nodules, but the dominant strain changed to B. japonicum in the
Matsue and Fukagawa locations, suggesting that the temperature-dependent infection
would lead to the nodule dominancy of B. elkanii in the Miyazaki location. In the Matsue
location, because the nodule composition partially changed in the Fukagawa location,
both factors would be involved in the determination of the nodule composition. In the
second year, BeL7 found to be increased in all soils and locations, which might be due to
the little higher temperature during one month after sowing in the second year.
Growth and competitive infection behaviors of two sets of Bradyrhizobium spp.
strains were examined at different temperatures to explain the strain-specific soybean
nodulation in Fukagawa and Miyazaki by using the Bradyrhizobium spp. strains isolated
from the corresponding soils and locations. Each set consisting of three strains was as
follows: B. japonicum Hh 16-9 (Bj11-1), B. japonicum Hh 16-25 (Bj11-2) and B. elkanii Hk
16-7 (BeL7); B. japonicum Kh 16-43 (Bj10J-2), B. japonicum Kh 16-64 (Bj10J-4) and B.
elkanii Kh 16-7 (BeL7), which were isolated from the soybean nodules cultivated in the
Fukagawa and Miyazaki soils, respectively. The growth of each strain was evaluated in
yeast mannitol (YM) liquid medium at 15, 20, 25, 30 and 35 °C with shaking at 125 rpm
for one week while measuring their OD660 daily. In the competitive infection experiment,
each set of the strains was inoculated in sterilized vermiculite followed by sowing surface
sterilized soybean seeds, and they were cultivated at 20/18 °C and 30/28 °C in 16/8 h
(day/night) cycle in a phytotron for three weeks. After three weeks plant length and
weight of shoot and root were measured, and number of nodules were counted. Then
nodule composition of the inoculated strains was examined using randomly selected ten
nodules per plant. The nodule compositions were determined based on the partial
16S-23R rRNA ITS gene sequence of the DNA extracted from the nodules.
The optimum growth temperatures were at 15–20°C for all B. japonicum strains,
while they were at 25–35 °C for all B. elkanii strains. The shoot and root lengths, and the
shoot and root weights were significantly higher at 30/28 °C than 20/18 °C in all
treatments except for the root lengths of the soybeans inoculated with the Miyazaki
strains. While the nodule numbers were not significantly different between the different
temperature conditions. In the Fukagawa strains, Bj11-1 and BeL7 dominated in the
nodules at the low and high temperatures, respectively. In the Miyazaki strains,
BjS10J-2 and BeL7 dominated at the low and high temperatures, respectively.
In the Fukagawa soil, because B. elkanii BeL7, which has higher ability to grow
and nodulate than the B. japonicum strains (Bj11-1 and 2) at the high temperature, did
not appear in the Matsue and Miyazaki locations, it was suggested that the B. japonicum
strains preferably proliferated in the Fukagawa soil, and Bj11-1 nodulated more
preferably than Bj11-2. In the Miyazaki soil, B. elkanii BeL7 dominated in the nodules,
but the B. japonicum strains BjS10J-2 and BjS10J-4 increased in the Fukagawa and
Matsue locations, suggesting that both B. japonicum and B. elkanii proliferate in the
Miyazaki soil, and B. elkanii (BeL7) dominated in nodules due to their
temperature-dependent infection.

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