Genetical and physiological characterization of nodule-derived bacteria for practical application as biofertilizers in leguminous crop cultivation in Bangladesh and Japan

概要

（別紙様式 14）
2020 年 8 月 19 日

論文の内容の要約
氏

Md Firoz Mortuza

名

学位の種類

博士（農学）

学府又は研究科・専攻

連合農学研究科

指導を受けた大学

東京農工大学

学位論文名

Genetical and physiological characterization of nodule-derived
bacteria

for

専攻生物生産科学

practical

application

as

biofertilizers

in

leguminous crop cultivation in Bangladesh and Japan
【論文の内容の要約】
Background: With the expected rise in worldwide population, there is an unavoidable

and momentous challenge to meet the necessities of the large population. To meet the
huge demand of food supply, utilization of chemical fertilizers in agriculture to produce
high amount of crops simultaneously damaging the environment by polluting air, water,
and soil to a great extent and causing harmful impacts on living organisms and
facilitating adverse climate change. Considering all the adverse effects of persistent use
of chemical fertilizers and significant capability of biofertilizer to grow healthy food
significantly with long-term sustainability in an eco-friendly manner, it is a timely
approach to search, identify and utilize potential microorganism as biofertilizer.
Methods: After collection of samples from different locations of Bangladesh and Japan,

many bacteria have been isolated from the samples. To achieve fundamental
understanding on the diversity and taxonomic identity, phylogenetical characterization
of isolated bacterial strains with the 16S rRNA, 16S-23S ITS, 23S rRNA, nifH, nodD1,
and gyrB genes were performed. In order to assess the stress tolerance capability of the
isolates and to find out suitable stress tolerant bacteria, pH, salinity and temperature
tolerance tests were performed. Pot experiments were performed to determine key
symbiotic properties of these bacteria and to evaluate their N2 fixing capability by
measuring ARA and other relevant parameters.
Analysis/Results: In case of Bangladeshi samples, 84 bacteria have been isolated and

characterized from root nodules of soybean plants grown in soil samples of 11
districts/locations. Phylogenetic analysis of the 16S rRNA gene sequence revealed
unpredictably diverse array of both nodule-forming and endosymbiotic bacteria
belonging

to

Bradyrhizobium

and

Methylobacterium

of

Alpha-proteobacteria,

Pandoraea of Beta-proteobacteria, Stenotrophomonas of Gamma-proteobacteria,
Bacillus of Firmicutes and Leifsonia of Actinobacteria phylum/class to be present
among the isolated bacteria. Unsurprisingly, most of the isolates (65 out of 84) showed
phylogenetic resemblance with the genus Bradyrhizobium; 45 isolates showed close
relationship with Bradyrhizobium diazoefficiens and Bradyrhizobium japonicum, 16
with Bradyrhizobium liaoningense and Bradyrhizobium yuanmingense, and four (4)
with Bradyrhizobium elkanii. Sequence analysis of the nifH gene demonstrated that
except the four isolates which showed the 16S rRNA gene resemblance with B. elkanii
and two isolates out of 16 of B. yuanmingense, all other isolates showed the nifH gene
similarity with B. diazoefficiens. In case of the nodD1 gene phylogeny, almost similar
results like the nifH gene tree with some discrepancies were observed and most of the
isolates - 70 in number - clustered in the same branch with B. diazoefficiens. Isolates
having similarity with B. yuanmingense, Methylobacterium, Pandoraea, Leifsonia, and

Stenotrophomonas (the nodD1 gene sequence was unconfirmed for Stenotrophomonas)
in the 16S rRNA based tree showed to possess the symbiotic genes (nifH and nodD1)
similar to B. diazoefficiens. B. yuanmingense having the symbiotic genes analogous to B.

diazoefficiens could be due to vertical gene transfer; whereas, the unconventional
existence of the symbiotic genes in isolates showing phylogenetic similarity with strains
of genera Methylobacterium, Pandoraea, Leifsonia, and Stenotrophomonas, could be the
outcome of lateral or horizontal gene transfer. Multigene phylogenetic analysis was
performed by concatenating the almost complete 16S rRNA gene, full-length of the
16S-23S Internal Transcribed Spacer (ITS) region, the partial 23S rRNA gene and the

gyrB gene sequences and displayed almost similar phylogeny like the 16S rRNA
gene-based tree. Twenty-one isolates were selected as representative strains of all
locations and bacterial types and were inoculated along with B. diazoefficiens USDA110
in two soybean varieties; Glycine max cv. Enrei and Glycine max cv. Binasoybean-3. One
of the Methylobacterium isolates, Dinaj-P1-S1-PS1/71’, did not form nodules in both the
soybean varieties; however, it produced plant shoot length (73.83±13.66 cm)
significantly higher than B. diazoefficiens USDA110 (38.07±4.24 cm) and biomass
production 11% in Enrei variety, therefore, single inoculation or co-inoculation of this
isolate with suitable nodule producing rhizobia may enhance soybean growth at a
greater extent. In regard of Binasoybean-3, Tkg-P2-B1-S1/68’ isolate belonging to B.

diazoefficiens, yielded shoot length (17.37±1.01 cm) significantly higher than B.
diazoefficiens USDA110 (13.57±1.12 cm) and produced 10% enhancement of biomass
production. Bhola-P2-B2-S1/51' isolate, having similarity with B. liaoningense, was the
only isolate which showed significantly higher acetylene reduction assay (ARA) activity

(in µmol/h/g nodule dry weight) in both Enrei and Binasoybean-3 variety than B.

diazoefficiens USDA110 and increased plant biomass production of 9% in case of
Binasoybean-3 variety. This isolate could grow at 1%-4% NaCl concentration, pH 4.5-10
and could survive at 45 °C making the isolate a potential candidate as biofertilizer for
soybean in tropical regions.
In case of Japanese samples, multiphase characterization of 20 bacteria derived from
the root nodules of V. riukiuensis grown in Ishigaki and Iriomote Islands of Japan was
performed. Multigene phylogenetic analysis of housekeeping genes based on the 16S
rRNA gene sequences, the 16S-23S rRNA gene Internal Transcribed Spacer (ITS) and
the 23S rRNA gene sequences identified three main groups closely similar to B.

japonicum, B. elkanii and B. jicamae family. However, analysis of the symbiotic nifH
and nodD1 genes and their phylogenetic trees showed similar topology, having only few
discrepancies in comparison to the housekeeping gene phylogeny. Interestingly, for some
of the isolates having similarity with B. elkanii, growth was observed at 40 °C, which
exceed the highest record for B. elkanii to the best of our knowledge. All the isolates
were observed to have the capability of forming root nodules and fix nitrogen in their
original host plant V. riukiuensis and two other crops: soybean and mungbean. Most of
the isolates showed similar or higher nitrogen-fixing capability in comparison with B.

diazoefficiens USDA110 in V. riukiuensis and V. radiata (mungbean), and Iri 5/6 in V.
riukiuensis, Iri 5/12 in soybean and Ishi 7/2 in mungbean showed highest ARA (in
µmol/h/g nodule dry weight) activity, which was significantly higher than B.

diazoefficiens USDA110. Furthermore, six isolates attained higher soybean biomass
production compared with B. diazoefficiens USDA110, suggesting high symbiotic
compatibility with soybean. Among them, Iri 5/7 of B. elkanii group contributed 29%
higher soybean biomass production than B. diazoefficiens USDA110 and could grow at
40 °C, hence it could be a promising soybean inoculant in the tropics.
Conclusion: In conclusion, it can be stated that the present study delivers the first

genetical and physiological characterization of soybean root nodule bacteria isolated
from soil samples of Bangladesh, and Vigna riukiuensis root nodule bacteria of Ishigaki
and Iriomote islands of Japan. Unpredictably diverse array of both nodule-forming and
endosymbiotic

bacteria

belonging

to

Alpha-proteobacteria

Beta-proteobacteria,

Gamma-proteobacteria, Firmicutes and Actinobacteria phylum/class were found to be
present

among

the

Bangladeshi

and

Japanese

isolates.

Bhola-P2-B2-S1/51',

Dinaj-P1-S1-PS1/71’, Tkg-P2-B1-S1/68’ isolates of Bangladesh and Iri 5/7 isolate of
Japan showed greater performance in plant growth and stress tolerance in comparison
to B. diazoefficiens USDA110 making these isolates suitable candidate as biofertilizer.