Microb. Resour. Syst. 35
June
（1）
：3
2019
─ 11, 2019Vol. 35, No. 1

Isolation and identification of nitrogen-fixing bacteria
associated with Dioscorea alata L. andDioscorea esculenta L.
Michel Ouyabe1)＊, Hidehiko Kikuno3), Naoto Tanaka2),
Pachakkil Babil1) and Hironobu Shiwachi1)
Faculty of International Agriculture and Food Studies, 2)Faculty of Life Sciences,
Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan3)
Miyako Subtropical Training and Research Farm, Tokyo University of Agriculture72-2 Gusukubefukuzato, Miyakojima-shi, Okinawa, 906-0103, Japan
1)

Yams are important tuber crops in the tropics. Nitrogen is one of the limiting factors in yam production. Yamaccessions that can rely on biological nitrogen fixation must be identified if we are to achieve efficient and sustainableyam production. The aim of this study was to isolate and identify the endophytic diazotrophic bacteria of 19 accessionsof two yam species. Forty-one endophytic bacteria were isolated from surface-sterilized roots, stems and leaves of yam
accessions grown in nutrient-poor subsoil for 160 days. The 16S rRNA gene sequencing showed that the 41 isolates weregrouped into 18 genera including Bacillus, Bradyrhizobium, Devosia, Ensifer, Mycobacterium, Neorhizobium, Paenibacillus,
Pseudoxanthomonas, Rhizobium and Xanthomonas. Most endophytes showed a positive response to nitrogenase activity
and were found in the stems (21) and the roots (14), while only six were found in the leaves. In Dioscorea esculenta, theacetylene reduction assay (ARA) values ranged from 4.1 to 57.4 nmol tube/24 h and from 4.1 to 164 nmol tube/24 h in
Dioscorea alata. In addition, the endophyte species were different among yam species and among yam accessions andwere affected by fertilization. These results indicate that the endophytic diazotrophic bacteria of yams are diverse.

Key words: Diazotroph, Endophytes, Nitrogenase, Yam

INTRODUCTION

2015), sugarcane (Magnani et al., 2013; Muangthong
et al., 2015) and sweet potato (Asis & Adachi, 2005).
However, there is a lack of information on endophytic diazotrophs in tropical plants (Suhandono et al.,2016).
Yams (Dioscorea spp.) are important tuber crops
grown throughout all the tropical zones, with thehighest fresh tuber production in Africa. In West
Africa more than 60 million people depend on thiscrop for their food (Asiedu & Sartie, 2010). In its
traditional growing systems in Africa, yams are cultivated without or with very little application of mineral fertilizers (e.g., N). In Asian countries such as
Japan and Myanmar where yams are also cultivated,little attention has been given to this crop.
Although cultivated as the dominant staple foodcrop in Papua New Guinea, Dioscorea esculenta, forexample, is usually grown in poor soils with low fertilizer input (O’Sullivan & Ernest, 2007). Trials on
the mineral fertilization of yams showed that several
genotypes do not respond to mineral N application(Diby et al., 2009; Ettien et al., 2014). Recently, nitro-

Nitrogen (N) is of great importance to agriculture
as it is required in larger amounts than other nutrients and is involved in vital biological processes.
Inappropriate use of N fertilizers results in environmental problems (e.g., the eutrophication of soils andwaters, climate change). The production of N fertilizers is unsustainable, and so the need to provide
plants with biologically fixed N is being increasingly
investigated. Plant endophytic bacteria are defined
as bacteria that live inside plant tissues withoutcausing visible disease symptoms. Diazotrophic bacteria are endophytic bacteria capable of biologicalnitrogen fixation (BNF) in both legumes and nonleguminous plants (Iniguez et al., 2004). BNF has
been documented for several important crops,
including rice (Mbai et al., 2013; Ferrando & Scavino,Corresponding author＊

E-mail: 46316401@nodai.ac.jp
Accepted: December 26, 2018
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Nitrogen-fixing bacteria of yams

Ouyabe et al.

gen-fixing bacteria associated with yams werereported for the accession E-2 of D. esculenta by
Rezaei et al. (2017). Additionally, Takada et al. (2017)
also reported that the accession A-19 of the wateryam (D. alata) grows in non-fertile soil and absorbs
nitrogen from the air. These reports indicate that
nitrogen-fixing bacteria may contribute considerably
to yam growth. However, in several studies conducted with different crops, it has been reported
that communities of endophytic bacteria varydepending on plant genotype (Elbeltagy et al., 2001),
suggesting an endophytic bacteria-host plant specificity. This study was designed to assess the nitrogen-fixing bacterial population associated with different accessions belonging to Dioscorea alata andDioscorea esculenta.

(DAP). The characteristics of this soil are given in
Takada et al. (2017). Nineteen yam accessions were
tested in this study including the accessions A-19
(Takada et al., 2017) and E-2 (Rezaei et al., 2017) asshown in Table 1. These accessions, with different
origins, are all maintained by TUA. The experimental design was a completely randomized bloc with
one repetition for the accessions.
Preparation of plant samples
Endophytic nitrogen-fixing bacteria in yams wereextracted at 160 DAP using the culture-dependent
method. Leaves, stems and roots were tested todetermine whether they were harboring nitrogenfixing bacteria. Three to five widely open andhealthy leaves without petioles were collected fromthe base, middle and top of the plant and thoroughly
washed with tap water to remove dust and soil particles. The leaves were cut into pieces of about 2×
2 cm2. The same procedure was followed whenselecting stems. The stems were cut into segmentsof about 2-3 cm long. The roots were washed withtap water to remove all attached soil particles andthen placed on paper tissues. About 2 g of eachsample were used in the surface sterilization process.

MATERIALS AND METHODS

A pot experiment was conducted from May to
November 2016 on 19 accessions belonging to two
species of yam (Dioscorea alata and Dioscorea escu︲
lenta) on Miyako Island, Tokyo University ofAgriculture (TUA) Miyako Farm, Okinawa, Japan (N
24° 70′, E 125° 28′). The samples were planted in 5 lpots in a greenhouse on May 30th, 2016. Each pot
was filled with 3 kg of nutrient-poor subsoil collectedon Miyako Island. The treatments consisted ofnitrogen application and control without nitrogen
application. Urea was used as a mineral source ofnitrogen and was applied 60 days after planting

Table 1

Surface sterilization
Samples were washed with two liters of distilledwater containing 2 drops of TWEEN® 20 (about 1 ml)

List of accessions used in the study on nitrogen-fixing bacteria associated with yam

Accessions
Yam species
code No.

OriginAccessions
Yam species
code No.

A-4

D. alata

Indonesia

A-68

A-16

D. alata

Nepal

A-73

A-17

D. alata

Kochi, Japan

A-74

A-18

D. alata

Okinawa, Japan

A-86

A-19
A-23
A-44
A-58
A-61

D. alata
D. alata
D. alata
D. alata
D. alata

Palau
Papua New Guinea
Keelung, Taïwan
Chiayi, Taïwan
Kagoshima, Japan

A-112
A-116
E-1
E-2
E-3

A-62

D. alata

Pyin Oo Lwin,
Mandalay, Myanmar
─4─

D. alata

OriginKagoshima University,
unknown
D. alata
Myitkyina, Kachin state,
MyanmarD. alata
Pyin Oo Lwin, Mandalay
division, Myanmar
D. alata
Kutkhaing, Shan state,
MyanmarD. alata
Okinawa, Japan
D. alata
Okinawa, JapanD. esculenta Lashio, Myanmar
D. esculenta Okinawa
D. esculenta Hateruma island, Okinawa,JapanMicrob. Resour. Syst. June 2019Vol. 35, No. 1

to remove any epiphytes. During surface sterilization, the plant samples were immersed successively
in a solution of 70% ethanol for 5 min, and 3% sodium hypochlorite (NaClO) for 10 min. The samples
were then transferred to a clean bench, rinsed 4times with sterile distilled water, and macerated
with a sterilized pestle and mortar with 10 ml ofautoclaved normal saline (0.9% NaCl). One milliliterof the macerate was mixed with 1 ml of 30% glycerol in Eppendorf tubes and stored at -15℃ for bacteria isolation and identification.

bacteria, an ARA was conducted on randomly
selected pure bacterial colonies. A semi-solid N-free
MR medium was prepared, and 4 ml was introduced
into 35-ml glass tubes that were closed with foam
stoppers. One loop of the bacterial isolates was
injected into the tubes and kept at 30℃ for 9 days.
The foam stoppers were replaced with rubber stoppers, and an atmosphere containing 10% acetylene
was created in the tubes by removing 3 ml air andreplacing it with an equal volume of acetylene gas.
The ethylene concentration was measured 24 h later
by injecting 1 ml of the atmosphere into a gas chromatograph equipped with a flame ionization detector (FID) and N Porapak column.

Isolation of bacterial colonies
Nitrogen-free modified Rennie (MR) medium(Elbeltagy et al., 2001) was used as the growing
medium. The MR medium was prepared from solutions A and B. Solution A (300 ml) consisted of 0.2 g
MgSO4・7H2O, 3 ml CaCl2 (0.2 g in 10 ml water), and
5 ml of 0.5% bromothymol blue (0.25 g in 50 ml 0.2 M
KOH). The pH was adjusted to 6.8 with 0.1 M HCl.
Solution B (700 ml) was made with 0.8 g K2HPO4,
0.2 g KH2PO4, 0.1 g NaCl, 5.0 g sucrose, 3.0 g mannitol, 2.0 g DL-malic acid, 100 mg Yeast extract (Difco),
1.25 ml Na2MoO4・2H2O (0.2 g in 10 ml water) and
1 ml NaFe(III)-EDTA (0.28 g in 10 ml water). The
pH was adjusted to 6.8 with 5 M KOH. A solid MR
growing medium was prepared with 20 g gellan
gum. As with as solution A, solution B was added
to the solidifying powder and autoclaved at 121℃
for 20 min. Under a clean bench, the two solutions
were mixed and 1 ml each of filter-sterilized biotin
and para-aminobenzoic acid were added. Plant macerates were injected into 40-ml glass tubes containing 4 ml of semi-solid N-free MR growth medium
and closed with a foam stopper. The tubes werethen incubated at 30℃ for 7-10 days. In further
studies, tubes with formations of bacterial pellicles
were considered to contain at least one nitrogen-fixing bacterium. These bacterial pellicles werestreaked on a solid N-free MR medium and incubated under the same conditions as mentioned above.
On each plate, bacterial colonies of different shapes
and colors were considered to be different strains
and were separately cultured on a fresh solid N-free
MR medium until pure colonies were obtained.
Bacteria with weak growth were excluded from theanalysis.

Bacterial genomic analysis
Pure bacterial colonies were subjected to an analysis of their 16S rRNA genes. A bacterial cell suspension (20 μl) was treated with proteinase K (5 μl)
at 60℃ for 20 min and 95℃ for 5 min to disrupt cell
membranes. The 16S rRNA genes were amplified
from the extracted DNA template with the universal primers 9F (5’-GAGTTTGATCCTGGCT-3’) and
1541R (5’-AAGGAGGTGATCCAGCCGCA-3’). The
PCR reaction mixture consisted of 10x ExTaq buffer
5 μl, dNTP Mix 4 μl, 1 μl of each primer, distilledwater (nuclease-free) 37.75 μl and enzyme ExTaq
0.25 μl. PCR was performed as follows: 35 cycles ofdenaturation at 94℃ for 30 sec, annealing at 55℃ for
30 sec, extension at 72℃ for 90 sec and final extension at 72 ℃ for 2 min. The PCR products were
purified and the bacterial DNA fragments
sequenced by Macrogen Japan Corp. The obtained
sequences of 400 to 800 bp were used for a similarity search of the 16S rRNA fragments in the 16S
database of EzBioCloud (Yoon et al., 2017). The
accession numbers of the DNA sequences were submitted to the DNA Data Bank of Japan (DDBJ).

RESULTS

Endophytic diazotrophic bacteria isolated fromyams
Endophytic bacteria were isolated from roots,
stems and leaves. Urea-treated plants were compared with untreated plants (control). The number
of isolates was higher in urea-treated plants (21).
For both the control and the urea treatment, thebacteria were isolated from all the tested organs.
Seven isolates in the urea-treated plants belonged tothe class Bacilli, nine to the class Actinobacteria. The

Acetylene reduction assay (ARA)
To confirm the nitrogenase activity of the isolated
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Nitrogen-fixing bacteria of yams

Ouyabe et al.

classes Alphaproteobacteria and Betaproteobacteria
were represented by two isolates each, and one strain
belonged to the class Gammaproteobacteria. In thecontrol plants, the class Bacilli was dominant with 4
isolates, but the Actinobacteria and Betaproteobacteria
strains were not detected. Overall, 21, 14 and six
endophytic nitrogen-fixing bacteria were isolated
from the stems, roots and leaves of yams, respectively. The phylum Proteobacteria accounted for
56.1% followed by the phylum Firmicutes at 34.15%
and the phylum Actinobacteria at 9.76%. The genus
Bacillus accounted for 31.71% of total isolates, followed by the genus Rhizobium (24.39%). Forty-one
endophytes were isolated on a nitrogen-free growing
medium from yam plants (Tables 2 and 3).
In Dioscorea esculenta (Table 2), all tested accessions could harbor fast growing endophytic bacteria
on an N-free MR medium. In total 11 isolates
belonging to seven genera were found in this species.
Seven isolates belonged to the phylum Proteobacteria,
all being in the class Alphaproteobacteria. Two isolates belonged to the class Bacilli and two to theclass Actinobacteria. Three bacteria were isolated
from accession E-1 and three from accession E-2,
while six were found in E-3. The bacteria of thephylum Actinobacteria were not isolated from accession E-1. No bacterial isolates were found in theleaves, while only two isolates were found in theroots of accessions E-1 and E-2. In this study, aTable 2

greater number of isolates were recovered from thestems (83%).
In Dioscorea alata (Table 3), fast growing endophytic diazotrophic bacteria were not found in someof the tested accessions. No bacteria were identified
from accessions A-16, A-18, A-19, A-62 and A-74. In
accessions from which bacteria were isolated andidentified, most of the isolates in all of the tested
organs were from urea-treated plants. Roots andstems harbored the same number of bacteria (12).
Five strains each were isolated from A-17 and
A-112, and four strains each were found in A-23 andA-86. In A-86 and A-112, all the isolates were fromurea-treated plants, while in A-17, all the bacteria
were from the control plants.
Acetylene reduction assay
All the randomly selected endophytic bacteria
exhibited positive ARA (Tables 2 and 3). In D. escu︲
lenta, the amount of ethylene produced after 24hours of incubation ranged from 4.1 to 57.4 nmol
C2H4/tube/24 h. The highest nitrogenase activity
was 57.4 nmol C2H4 by Bradyrhizobium sp., isolate
E1T0R_Y6, found in the root of E-1 under unfertilized conditions. In D. alata, the ARA values ranged
from 4.1 to 164 nmol C2H4, the highest value being in
isolate A23T1R_Y40. In D. alata, isolates showing ahigh ARA value were found in urea-treated plants.

Population of diazotrophic bacteria isolated from accessions of
Isolatea

E1T0R_Y6 (LC373043)
E2T0R_Y14 (LC373045)
E3T1S_Y82 (LC373077)
E3T1S_Y17 (LC373047)
E3T1S_Y83 (LC373078)
E3T1S_Y84 (LC373079)
E2T1S_Y5 (LC373042)
E2T0S_Y16 (LC373046)
E3T0S_Y23 (LC373048)
E1T1S_Y73 (LC373072)
E3T1S_Y80 (LC373076)

OriginOrgan Accession Treatment

Close to: (%)

ARAb

Root

E-1
E-2

T0
T0

Bradyrhizobium lupini (100)Bacillus altitudinis (99.4)

57.4
NT

Stem

E-3
E-3
E-3
E-3
E-2
E-2
E-3
E-1
E-3

T1
T1
T1
T1
T1
T0
T0
T1
T1

Curtobacterium citreum (99.7)
Sphingomonas sp. (98.6)
Neorhizobium huautlense (99.8)
Bacillus aryabhattai (100)Kocuria sp. (95.1)
Rhizobium multihospitium (99.0)
Rhizobium sp. (98.3)
Rhizobium multihospitium (100)
Neorhizobium huautlense (99.9)

4.1
8.2
8.2
8.2
NT
8.2
8.2
4.1
8.2

Bacterial isolates are followed by DNA sequences accession number submitted to the DNA Data
Bank of Japan (DDBJ). Numbers in parenthesis in related species represent the sequence similarity percentage.
b
ARA denotes the acetylene reduction assay value, expressed in nmole C2H4/tube/24 h. Plants
were treated with urea (T1) as source of mineral nitrogen or without urea (T0) as a control. NT:
not tested.
a─6─

Microb. Resour. Syst. June 2019

Table 3Vol. 35, No. 1

Related bacterial genus of diazotrophic bacteria isolated from accessions of
Isolatea

OriginClose to: (%)

Organ Accession Treatment

ARAb

A86T1R_Y60 (LC373061)
A68T1R_Y54 (LC373056)
A58T1R_Y51 (LC373054)
A23T1R_Y55 (LC373057)
A112T1R_Y65 (LC373065)
A23T1R_Y40 (LC373052)
Root
A17T0R_Y78 (LC373075)
A23T0R_Y39 (LC373051)
A17T0R_Y27 (LC373049)
A68T1R_Y52 (LC373055)
A112T1R_67 (LC373067)
A112T1R_Y66 (LC373066)

A-86
A-68
A-58
A-23
A-112
A-23
A-17
A-23
A-17
A-68
A-112
A-112

T1
T1
T1
T1
T1
T1
T0
T0
T0
T1
T1
T1

Bacillus altitudinis (100)
Achromobacter xylosoxidans (100)Devosia yakushimensis (99.4)
Rhizobium phenanthrenilyticum (99.1)
Sphingomonas sp. (98.2)
Ensifer sp. (98.3)
Mesorhizobium sp. (98.3)
Paenibacillus panacisoli (98.9)
Nitratireductor sp. (98.5)
Pseudoxanthomonas indica (99.4)
Rhizobium massiliae (100)Rhizobium multihospitium (100)

65.6
NT
123
NT
12.3
164
NT
12.3
   8.2
NT
12.3   4.1

A86T1S_Y61 (LC373062)
A61T1S_Y71 (LC373070)
A4T1S_Y87 (LC373080)
A17T0S_Y77 (LC373074)
A44T0S_Y94 (LC373082)
A112T1S_Y63 (LC373064)
Stem
A17T0S_Y77 (LC373074)
A86T1S_Y72 (LC373071)
A23T1S_Y37 (LC373050)
A116T1S_Y46 (LC373053)
A61T0S_Y69 (LC373069)
A17T0S_Y76 (LC373073)

A-86
A-61
A-4
A-17
A-44
A-112
A-17
A-86
A-23
A-116
A-61
A-17

T1
T1
T1
T0
T0
T1
T1
T1
T1
T1
T0
T0

Bacillus aryabhattai (100)Bacillus aryabhattai (100)Bacillus aryabhattai (100)Enterobacter cloacae (100)Bacillus aryabhattai (100)Burkholderia contaminans (100)Enterobacter cloacae (100)Mycobacterium cosmeticum (100)Rhizobium sp. (98.5)
Rhizobium sp. (97.8)
Rhizobium multihospitium (100)
Xanthomonas sacchari (99.2)   8.2   8.2   8.2NT   8.2
NT
NT
NT
NT
41
12.3
NT

A73T1L_Y58 (LC373059)
A86T1L_Y59 (LC373060)
A61T0L_Y68 (LC373068)
A4T1L_Y90 (LC373081)
A73T0L_Y57 (LC373058)
A112T1L_Y62 (LC373063)

A-73
A-86
A-61
A-4
A-73
A-112

T1
T1
T0
T1
T0
T1

Bacillus aryabhattai (99.8)
Bacillus aryabhattai (100)Bacillus aryabhattai (100)Bacillus arryabhattai (100)Bacillus subtilis (100)Curtobacterium oceanosedimentum (100)   8.2   8.2   8.2   8.2   8.216.4

Leaf

Bacterial isolates are followed by DNA sequences accession number submitted to the DNA Data Bank ofJapan (DDBJ). Numbers in parenthesis in related species represent the sequence similarity percentage.
b
ARA denotes the acetylene reduction assay value, expressed in nmole C2H4/tube/24 h. Plants were treatedwith urea (T1) as source of mineral nitrogen or without urea (T0) as a control. NT: not tested.
a

DISCUSSIONS

isolated from yam accessions. The class
Alphaproteobacteria was the dominant class andincluded the genera Bradyrhizobium (1), Devosia (1),
Ensifer (1), Mesorhizobium (1), Neorhizobium (2),
Nitratireductor (1), Rhizobium (9), and Sphingomonas(2). Our finding was in line with previous studies
showing that strains of the class Alphaproteobacteria
are found in a large number as nitrogen-fixing bacteria in soils and tissues of legumes and non-legumes(Tsoy et al., 2016). We isolated Ensifer sp. A23T1R_
Y40 with the highest acetylene reduction. Somestrains of this genus such as Ensifer medicae arecapable of fixing biological nitrogen (Reeve et al.,2010; Li et al., 2016). There have been few reports

The objective of this study was to assess the nitrogen-fixing bacteria associated with several yam
accessions unlike in previous studies. In our study,
endophytic diazotrophs could not be isolated fromfive yam accessions; all in D. alata, including A-19.
The reaction time during the surface-sterilization
step was longer than in other studies (Muangthong
et al., 2015), resulting in possible damage to plant tissue and endophytic bacterial cells. This could also
explain why only the accessions of D. alata, whichhave softer tissues than D. esculenta, were affectedby the strong surface-sterilization treatment.
Several strains belonging to different genera were─7─Nitrogen-fixing bacteria of yams

Ouyabe et al.

on the presence of this genus in non-legumes.
Recently, one strain was isolated from the roots ofrice (Zhang et al., 2010). To our knowledge, this is
the first study reporting the genus Ensifer as endophytic nitrogen-fixing bacteria in a tuber crop.
The bacterial classes Alphaproteobacteria,
Betaproteobacteria and Gammaproteobacteria havebeen reported as nitrogen-fixing bacteria in plants
and soils (Taule et al., 2012). In our study, we identified strains of the genera Achromobacter andBurkholderia, and Enterobacter, Pseudoxanthomonas
and Xanthomonas, in the classes Betaproteobacteria
and Gammaproteobacteria, respectively. A strain,
A68T1R _ Y52 s h o w i n g 99.38% s i m i l a r i t y w i t h
Pseudoxanthomonas indica was isolated in thisstudy. Grapevine roots have been reported to harbor strains of the genus Pseudoxanthomonas, which
possesses plant growth-promoting characteristics(Marasco et al., 2013). However, evidence of nitrogen
fixation by this genus has yet to be reported. In
this study, the isolate A68T1R_Y52 grew quickly on
an N-free MR medium.
In the phylum Firmicutes, thirteen strains wereisolated and identified as being closely related to
Bacillus spp. (12) and Peanibacillus panacisoli (1).
Several strains belonging to these two genera wereshown to be capable of biological nitrogen-fixation in
non-legumes (Ding et al., 2005). Grady et al. (2016)
reported that strains of Paenibacillus species play
important roles, including hormone production andphosphate solubilization. Several strains of thisgenus were identified as nitrogen-fixing bacteria(Xie et al., 2014). This is the first report on the ability of P. panacisoli to fix atmospheric nitrogen as
plant endophytic bacteria. Different strains ofBacillus spp. were isolated from tissues of several
crops such as rice (Mano et al., 2006, 2007; Mbai etal., 2013) and sugarcane (Magnani et al., 2010) withplant growth-promoting characteristics.
Numerous strains belonged to the phylum
Actinobacteria have been involved in plant growthpromoting activity, including nitrogen fixation, phosphate solubilization and phytohormones production(Sathya et al., 2017), with the genus Mycobacterium
as one of the most common isolates., Kocuria sp.
E2T1S_Y5 was isolated in the stem of D. esculenta
E-2. Important crops such as rice have been shownto harbor strains belonging to the genus Kocuria intheir seeds (Kaga et al., 2009). These bacteria werealso identified in the Jatropha (Jathropha curcas)

plant as endophytic bacteria (Madhaiyan et al., 2015).
To our knowledge, this study is the first report on
the potential nitrogen fixation of Kocuria sp.
The application of mineral N in the form of urea
did not reduce the number of endophytic nitrogenfixing bacteria as previously reported (Lin et al.,2012). The amount of urea applied in this study was
10 times less than the amount recommended foryams in Okinawa prefecture (Japan). Although theeffect of nitrogen application on the dynamics ofendophytic bacteria of yams is not yet known, theamount of urea applied in this study (using
N-deficient soil) could provide the bacteria with anadequate amount of nutrient N to realize a better
growth and colonization ability. All the isolates tested in this study could grow satisfactorily on an
N-free medium, supporting their ability to fix nitrogen from air. Representative isolates were selected
and tested for acetylene reduction, and they could
all reduce acetylene. Further studies are needed toevaluate the potential of these isolates as plant
growth-promoting bacteria through biological nitrogen fixation in yams. This study revealed thestrong effect of plant genotype on the number ofendophytic diazotrophs found in yams. Several
studies have reported that plant roots harbor more
endophytic bacteria than stems and few bacteria arefound in leaves (Gyaneshwar et al., 2001; Koomnok etal., 2007) due to the fact that most endophytic bacteria in plants are recruited from the surrounding soil.
However, our study showed that in yams, the plant
genotype has a strong effect. Significant differenceswere observed among accessions of D. esculenta.
Thus, no fast-growing endophytic diazotrophs were
found in the roots of accession E-3, in which all isolated bacteria were in the stems. An investigationof the physiological and molecular factors involvedin bacteria movements within yam plant organs isneeded. Overall, accessions of D. esculenta harboredmost endophytic bacteria in the upper parts (stems
and leaves). In D. alata, there was no difference
between the number of diazotrophs in the roots andthe stems. This finding was in line with that ofKoomnok et al. (2007) who reported that depending
on rice genotype, a similar number of nitrogen-fixing
bacteria was found in the leaves, stems and roots asalso reported in sugarcane by Muangthong et al.
(2015).

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Microb. Resour. Syst. June 2019Vol. 35, No. 1

ACKNOWLEDGEMENTS

pneumoniae 342. Mol. Plant Microbe Interact. 17:
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Koomnok, C., Teaumroong, N., Rerkasem, B. &Lumyong, S. 2007. Diazotroph endophytic bacteria
in cultivated and wild rice in Thailand. Sci. Asia
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Our gratitude goes to the Ministry of Education,
Culture, Sports, Science, and Technology (MEXT)
Japan who granted us a scholarship for this study.
We also thank Tokyo University of Agriculture forproviding funding for all materials and trips.

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Microb. Resour. Syst. June 2019Vol. 35, No. 1

ダイジョ系統とトゲイモ系統に関連する窒素固定細菌の分離と同定
ミシエル・オウヤベ 1），菊野日出彦 3），田中尚人 2），パチャキル・バビル 1），志和地弘信 1）
東京農業大学国際食料情報学部，2）同生命科学部，3）同宮古亜熱帯農場

1）

ヤムイモ（Dioscorea spp.）は熱帯地域などでは重要な作物である．近年，ヤムイモの生長には内生する窒素固定細菌が寄与している可能性が指摘され始めている．しかしヤムイモには多様な系統が存在するため，本研究ではダイジョ（Dioscorea
alata L.）の 16 系統とトゲイモ（Dioscorea esculenta L.）の 3 系統，併せて 2 種 19 系統のヤムイモを窒素施用と無施肥の条件で 160 日栽培し，茎，根，葉の部位に内生する窒素固定細菌の分離と 16S rRNA 遺伝子配列に基づく同定を行った．その結果，全部位から窒素固定細菌が併せて 41 株が分離された．分離株は Bacillus 属，Bradyrhizobium 属，Devosia 属，Ensifer
属，Mycobacterium 属，Neorhizobium 属，Paenibacillus 属，Pseudoxanthomonas 属，Rhizobium 属，Xanthomonas 属など
18 属と同定された．分離株の窒素固定能はアセチレン還元活性で評価し，トゲイモからの分離株は 4.1 から 57.4 nmol C2H4

tube/day/24 h，ダイジョからの分離株は 4.1 から 164 nmol C2H4 tube/day/24 h であり，試験した株はすべて固定能を有していた．そして，部位や系統の違い，施肥の有無による内生窒素固定細菌種が異なり，ヤムイモに内生する窒素固定細菌は多様であることが明らかとなった．

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