Advances in Microbiology, 2011, 1, 13-22
doi:10.4236/aim.2011.11003 Published Online December 2011 (http://www.SciRP.org/journal/aim)
13
Arbuscular Mycorrhizal Fungal Community Structure in
Soybean Roots: Comparison between Kanagawa and
Hokkaido, Japan
Katsunori Isobe1*, Kohei Maruyama1, Singo Nagai1, Masao Higo1, Tomiya Maekawa1,
Gaku Mizonobe2, Rhae A. Drijber3, Ryuichi Ishii1
1College of Bioresource Sciences, Nihon University, Fujisawa, Japan
2Hokkaido College, Senshu University, Bibai, Japan
3Department of Agronomy and Horticulture, University of Nebraska-Lincoln, Lincoln, USA
Received November 5, 2011; revised November 21, 2011; accepted December 10, 2011
Abstract
The objectives of this study were to determine arbuscular mycorrhizal fungi (AMF) community structure in
colonized roots of soybean cultivated from Kanagawa and Hokkaido in Japan and to relate the community
structure to environmental conditions, which included soil type, preceding crops, and soil chemical proper-
ties. The average number of AMF OTU (operational taxonomic unit) colonizing soybean roots collected
from Kanagawa and Hokkaido was 11.2 and 5.8, respectively, a significant difference. Moreover, AMF from
the family Gigasporasera was not identified in soybean roots collected from Hokkaido, suggesting that AMF
in the family Gigasporasea is absent or rare in the soybean fields of sampled in Hokkaido. We postulate that
the soil type, preceding crops or soil chemical properties are not the underlying factor differentiating AMF
community structure colonizing in soybean roots between Kanagawa and Hokkaido. Instead we conclude
that temperature and phosphate absorption coefficient are the determining factors of AMF OTU in this study.
Keywords: AM Fungi, Colonization, Community Structure, Soybean
1. Introduction
Arbuscular mycorrhizal fungi (AMF) are one of the most
important soil microorganisms, forming symbiotic asso-
ciations with terrestrial plant roots of most species. It is
well known that AMF improve the uptake of immobile
mineral nutrients such as phosphate, thereby benefiting
plant growth [1-3]. The extent of such benefit varies with
the soil environment, particularly available P content and
soil moisture. However, one of the most important fac-
tors in promoting host plant growth is an increase in rate
of colonization with AMF, which itself is strongly influ-
enced by AMF density in the soil. Therefore, to effec-
tively use AMF for crop cultivation, it is extremely im-
portant to clarify the density of AMF in upland field soil.
Isobe et al. [4] reported that AMF spore density was
higher in the soils with a higher phosphate absorption
coefficient, and negatively correlated with the available
phosphorus content. Moreover, the AMF colonization
rate of soybean in regions of Japan was largely influ-
enced by soil chemical properties [4]. AMF colonization
rate was positively correlated with AMF spore density,
and negatively with available soil phosphorus. In gener-
ally, AMF spore density is also assumed to be lower in
acidic and alkaline soils [5,6]. From these facts, we con-
sidered that the AMF spore density in field soil affected
by soil condition, for example soil chemical property.
More than 150 AMF species have been described
based on their spore morphology [7]. The individual
AMF species may differ in their growth response to dif-
ferent plant [8,9]. These functional differences underlie
the importance of clarifying which AMF species colo-
nize specific crops [10]. Consequently, the presence or
absence of AMF, as well as overall AMF community
structure or diversity has been shown to affect plant pro-
ductivity in agricultural ecosystems [11-13]. High AMF
diversity may result in more agricultural production pro-
vided that the co-existing AMF species can benefit the
crop under various stress conditions [14]. However, low
diversity is not necessarily disadvantageous if the few

K. ISOBE ET AL.
14
AMF species present are beneficial under a broad range
of agricultural and edaphic conditions [15].
Recently, molecular methods have been used in stud-
ies of AMF community structure in roots and soil [16,17].
Consequently, there are several reports of AMF commu-
nity structure in field soils and crop roots based on mo-
lecular approaches [18-22]. AMF community structure in
soils and/or roots has been shown to differ between geo-
graphic regions [23] and is responsive to various factors
including soil type, environmental conditions and host
plans [2,24-26]. Isobe et al. [4] reported that the coloni-
zation rate of the soybean roots at Hokkaido were lower
than that of root samples collected from Kanagawa. But,
in this paper, AMF community structure in soybean roots
collected from Hokkaido and Kanagawa did not investi-
gate. The objectives of this study were to investigate
arbuscular mycorrhizal fungi (AMF) community struc-
ture in colonized roots of soybean cultivated from Kana-
gawa and Hokkaido in Japan and to relate the community
structure to environmental conditions, which included
soil type, preceding crops, and soil chemical properties.
2. Material and Methods
2.1. Sampling Site and Sampling Method
Soybean (Glycine max L. Merr.) roots and rhizosphere
soils were sampled in August 2006 from six sites in
Kanagawa and four sites in Hokkaido, Japan. Each sam-
pling site in Kanagawa and Hokkaido was located within
a 40 km radius from Nihon University and Senshu Uni-
versity, respectively. Kanagawa (Fujisawa-city) and Hok-
kaido (Bibai-city) have a temperate climate with a mean
annual temperature of 15.9 and 7.1 C, and 1448 and 1156
mm annual precipitation, respectively. All sites are less
than 100 m in altitude. The size of each field was differ-
ent at each location, the smallest field was about 100 m2
(No. 6) and the biggest field was about 5000 m2 (No. 9).
Roots and soils were collected from the center of each
field. Roots were sampled from 15 randomly selected
plants and rhizosphere soil was collected within the cir-
cumference of the sampled roots, 15 cm in depth × 20
cm in diameter. Roots were divided into two subsamples
(about 100 roots of 1.5 cm length) and used for meas-
urement of AMF colonization rate and DNA extraction.
The variety of soybean was Enrei in Kanagawa, and Tsu-
rumusume or Iwaiguro in Hokkaido, and the sampling
time was at the flowering stage of soybean. Table 1
shows the sowing date of soybean, sampling date, variety
of soybean, soil group and preceding crops.
2.2. Analysis of Soil Chemical Property and
AMF Spore Number
A portion of the soil sample was air-dried for measure-
ment of chemical properties and the remainder was used
to determine the number of AMF spores. The pH (H2O),
phosphate absorption coefficient and available phosphate
were measured by the glass electrode method, the am-
monium phosphate method, and Bray 2 method, respec-
tively. AMF spores were recovered by wet sieving through
a 53 μm mesh followed by sucrose density gradient cen-
trifugation, and then were counted under a microscope.
Spore morphology was also recorded.
2.3. Analysis of AMF Colonization Rate in
Soybean Root
Soybean roots were stained with trypan blue and meas-
ured for AMF colonization rate by a grid crossing-point
method and the presence of hyphae, dendrophyses, and
cystidia were noted [27]. The size of grid was 5 mm
quarters; the count number of the crossing of grid and
root in one plant was lowest 200.
2.4. Analysis of AMF Community Structure
2.4.1. D NA Extrac ti o n from the Ro ots
Root samples were pulverized in a 2.0-mL tube contain-
ing five 2.0 mm zirconium balls using an MS-100 micro-
homogenizing system (Tomy Digital Biology Co., Ltd.,
Tokyo, Japan) at 4000 rpm for 1 min. The samples were
homogenized again at 4000 rpm for 1 min after the addi-
tion of 500 μL of 2 × hexadecyltrimethylammonium
bromide (CTAB) solution [2% CTAB, 0.1 M Tris-HCl
(pH 8.0), 20 mM EDTA, 1.4 M NaCl, and 0.5% β-mer-
captoethanol]. The homogenate was incubated in a block
heater at 65˚C for 1h. After adding 500 μL of chloroform
and isoamyl alcohol mixture (24:1, v:v), each tube was
vortexed and then centrifuged at 20,400 × g for 7 min.
The supernatant was transferred to 1.5-mL tube, and the
DNA was precipitated by adding an equal volume of
isopropyl alcohol then stored at –30˚C for 10 min. After
centrifugation at 5,800 × g at 4˚C for 10 min, the DNA
pellet was washed with 80% ethanol and resuspended in
120 μL of TE buffer solution [10 mM Tris-HCl (pH 8.0)
and 1 mM EDTA], and stored at -30°C before analysis.
2.4.2. M olecular An alysis
A portion of the LSU rDNA region of fungal DNA was
amplified using a nested PCR method [28]. The DNA
samples extracted from plant roots were diluted 20-fold
and used as PCR templates. Primers of LR1 and FLR2,
which are specific to fungi, were used to amplify the 5’-
end of the LSU rDNA region [29]. The method and PCR
conditions were the same as those described by Wu et al.
[30]. PCR was performed in 10-μL reaction mixtures
containing 1 μL template DNA, 1 μL of 10 × PCR buffer,

15 K. ISOBE ET AL.
0.2 mM of each dNTP, 0.3 μM of each primer, and 0.25
U of TaKaRa Taq DNA polymerase (Takara Shuzo Co.,
Tokyo, Japan) using a thermocycler (GeneAmp 9700,
Applied Biosystems). The PCR protocol was 1 cycle at
94˚C for 1 min; 29 cycles at 94˚C for 30 s, 54˚C for 30 s,
and 72˚C for 1 min; and 1 cycle at 94˚C for 30 s, 54˚C
for 30 s, and 72˚C for 10 min.
The first-round PCR products were diluted 100-fold
and used as templates for the second-round PCR using
nested primers FLR3 and FLR4 under the same PCR
conditions as described above [28,31]. The FLR3 primer
was labeled with Texas Red to generate 5’-end-labeled
products. The second-round PCR products (FLR3-4) were
separated by electrophoresis and analyzed for fragment
length polymorphism following Wu et al. [30]. The PCR
products (FLR3-4) were diluted appropriately with TE
buffer, denatured at 95˚C for 5 min, and loaded on 6%
sequencing gels (Long Ranger; Cambrex Bio Science
Rockland Inc., Rockland, USA) made with 1.2 × TBE
[0.1 M Tris, 3.0 mM EDTA, and 0.1 M boric acid] con-
taining 6.1 M urea. The samples were subjected to elec-
trophoresis in 0.6 × TBE on a DNA sequencing appara-
tus (SQ-5500; Hitachi Electronics Engineering Co., To-
kyo, Japan). DNA size standards were loaded at every
tenth lane on the gel. The length of each fragment was
estimated from the size standards using FRAGLYS 3.0
software (Hitachi Electronics).
The second-round FLR3-4 PCR products were re-am-
plified from the diluted first-round PCR products (LR1
to FLR2) and subcloned into pT7Blue using the Perfectly
Blunt Cloning Kit (Novagen Inc., Madison, WI) follow-
ing the manufacturer’s instructions. Colonies were ran-
domly selected. The plasmid DNA was extracted from
transformed E. coli cells for 5 min at 94˚C and used as a
template for PCR using the PCR conditions described
above. PCR was conducted using M13 forward and re-
verse primers (RPN 2337 and RPN 2338; Amersham
International plc., Buckinghamshire, England), and the
products were electrophoresed on agarose gels to con-
firm insertion of the FLR3-4 PCR fragments into the
plasmids. To sequence the inserts, a part of plasmids
containing FLR3-4 fragments were sequenced in both
directions using Texas Red M13 forward or T7 primers
by cycle sequencing using ThermoSequenase Pre-Mixed
Cycle Sequencing Kits (RPN 2444, Amersham Interna-
tional) following the manufacturer’s instructions and
were analyzed on a DNA sequencer (Hitachi Electronics).
The other plasmids were also sequenced in both direc-
tions using M13 forward or M13 Reverse primers by
cycle sequencing using a BigDye Terminator v3.1/1.1
Cycle Sequencing Kit (Applied Biosystems) following
the manufacturer’s instructions and were analyzed on a
DNA sequencer (ABI 3100, Applied Biosystems).
2.4.3. Data Analysi s
Species of AMF were inferred from sequence homolo-
gies with sequences recorded in the DNA Data Bank of
Japan (DDBJ, http://www.ddbj.nig.ac.jp/). Multiple ali-
gnments and neighbor-joining phylogenic trees were
constructed using MEGA 4 [32]; Mortierella verticillata
(accession no. AF157199) were used as out-groups.
2.5. Statistical Analysis
A one-way analysis of variance (ANOVA) followed by
Tukey’s multiple means test was done to compare soil
pH, phosphorus absorption coefficient, available phos-
phate content, AMF spore density, and AMF coloniza-
tion rate among sites at P < 0.05. Significant differences
in spore density, AMF colonization rate and number of
AMF OTU between Kanagawa and Hokkaido was de-
termined by t-test. The coefficient of correlation between
AMF spore density or colonization rate and soil chemical
properties (pH, phosphorus absorption coefficient, avai-
lable phosphate content) or AMF colonization rate were
also measured. Correlation analysis was done using Ka-
leida Graph ver.4.0 software.
3. Results
The pH of the collected soil ranged from 5.4 (No. 9) to
7.4 (No. 4). There was a significant difference in pH with
sampling site. The soil samples collected at sites 3, 4 and
5 had the highest pH, and the samples collected at sites 9
and 10 had a lower pH than the other samples. The soil
samples collected at sites 3, 4, 5 and 6 had the highest
pH, and the samples collected at 9 and 10 had a lower pH
than the other samples. The phosphorus absorption coef-
ficient of the collected soil ranged from 460 (No. 7) to
2270 (No. 5). There was a significant difference in phos-
phorus absorption coefficient with sampling site. The
soil samples collected at sites 5 and 6 had the highest
phosphorus absorption coefficient. The available phos-
phorus content ranged from 0.42g kg-1 (No. 8) to 0.07 g
kg-1 (No. 5) and there was a significant difference in
available phosphorus content with sampling site. The soil
sample collected at site 8 had the highest available phos-
phorus content, and the sample collected at site 5 had
lower available phosphorus content than the other sam-
ples. The AMF spore density differed with the sampling
site. The spore density was highest at sites 5 and 6, and
lowest at site 10. There was significant difference in
spore density between sites 5 and 6 and sites 1, 2, 7, 8
and 10. The colonization rate of soybean roots also var-
ied greatly among sites. The root samples at site 5 had
the highest colonization rate followed by those from sites
6 and 8. The colonization rate of the roots at site No. 3

K. ISOBE ET AL.
16
was lower than that of root samples collected from sites
1, 5-9 and 10 (Table 2).
The phylogenetic tree (Figure 1) contains AMF spe-
cies/clones from the Glomaceae, Acaulosporaceae, Gi-
gasporaceae, Diversisporaceae and Paraglomeraceae. LSU
rDNA gene sequences were partitioned into 25 clusters,
potentially yielding 25 OTU. The sequence identity within
the clusters ranged from 97.5% to 100%. Among the 25
OTU, 14 belonged to the Glomaceae, five to Gigaspo-
raceae, four to Acaulosporaceae, one to Diversisporaceae
and one to Paraglomeraceae. The AMF population was
heavily dominated by the Glomaceae. And OTU of Glo 1
belonged to the Glomaceae colonized soybean roots of
all sampling sites. OTU of Acaulosporaceae only colo-
nized soybean roots collected from site No. 1, 2 in
Kanagawa and site No. 9 in Hokkaido. And OTU of Di-
versisporaceae only colonized soybean roots collected
from site No. 1 in Kanagawa, and OTU of Paraglom-
eraceae only colonized soybean roots collected from site
No. 4 in Kanagawa and No. 9 in Hokkaido. AMF of Gi-
gasporaceae (Gig 1-3, Scu 1, 2) did not colonized soy-
bean roots collected from Hokkaido (No. 7-10). The
number of AMF OTUs were different among sampling
sites. In soybean roots collected from Kanagawa, the
number of AMF OTUs ranged from eight (No. 4) to 15
(No. 6). Four to seven OTUs were found in soybean
roots collected from Hokkaido (Figure 1, Table 3).
Table 4 shows the average AMF spore density in the
soil, AMF colonization rate, and number of AMF OTUs
colonizing soybean roots collected from Kanagawa (No.
1-6) and Hokkaido (No. 7-10). The average AMF spore
density collected from Kanagawa and Hokkaido was
4.5/g and 3.3/g, respectively. The average AMF coloni-
zation rate collected from Kanagawa and Hokkaido was
13.0% and 15.0%, respectively. And the average number
of AMF OTU in root collected from Kanagawa and
Hokkaido was 11.2 and 5.8, respectively. There were no
significant differences in spore density and colonization
rate between Kanagawa and Hokkaido. However, there
was a singnificant difference (p = 0.01) in the number of
AMF OTUs colonizing soybean roots between Kana-
gawa and Hokkaido. The average number of AMF OTUs
colonizing soybean roots collected from Kanagawa and
Hokkaido was 11.2 and 5.8, respectively. Table 5 shows
the correlation coefficient between AMF spore density in
field soil, AMF colonization rate and soil chemical prop-
erties (pH, phosphate absorption coefficient and avail-
able phosphorus content). AMF spore density was posi-
tively correlated (r = 0.751; p = 0.05) with the phosphate
absorption coefficient. However, the correlation coeffi-
cients of the AMF spore density with soil pH, available
phosphorus content and AMF colonization rate were not
significant. And the correlation coefficients of the AMF
colonization rate with soil pH, phosphate absorption co-
efficient and available phosphorus content were not sig-
nificant, too.
4. Discussion
4.1. AMF Spore Density and Colonization Rate
among Regions in Kanagawa and Hokkaido
We found that AMF spore density in soybean fields and
AMF colonization rate of soybean roots did not differ
between Kanagawa and Hokkaido (Table 4). The same
results were obtained by Isobe et al. [5], for other regions
of Japan. In the present study, AMF spore density and
colonization rate varied remarkably with sampling sate;
however, a highly positive correlation (r = 0.751*) was
observed between AMF spore density and the phosphate
absorption coefficient of the soil (Table 5) in agreement
with previous reports [4]. This suggests a higher abun-
dance of AMF spores in soils where the potential for P
fixation is high, e.g. volcanic ash soils. This is supported
by comparatively higher AMF spore density in andosols
(sites 5 and 6) and volcanogenous regosols (sites 8 and 9)
(Tables 1, 2). In soils with the high phosphate adsorption,
AMF play an important role in acquiring P for the crop.
Isobe et al. [4] reported that AMF colonization rate
was negatively correlated with the available phosphorus
Table 1. Soybean sowing date, sampling date, variety of soybean, soil group and preceding crop of each sampling site.
Prefecture Site No. Sowing date (M/D/Y) Sampling date (M/D/Y)Variety of soybeanSoil group Preceding crop
(Host plant of AMF or Non)
Kanagawa
Hokkaido
1
2
3
4
5
6
7
8
9
10
Jun./02/2006
Jun./02/2006
Jun./02/2006
Jun./02/2006
Jun./01/2006
Jun./01/2006
May/18/2006
May/17/2006
May/17/2006
Jul./11/2006
Aug./08/2006
Aug./08/2006
Aug./08/2006
Aug./08/2006
Aug./07/2006
Aug./07/2006
Aug./10/2006
Aug./10/2006
Aug./10/2006
Aug./16/2006
Enrei
Enrei
Enrei
Enrei
Enrei
Enrei
Tsurumusume
Iwaiguro
Iwaiguro
Iwaiguro
Brown lowland soil
Brown lowland soil
Brown lowland soil
Gray lowland soil
Andosol
Andosol
Gray lowland soil
Volcanogenous regosol
Volcanogenous regosol
Pseudogley soil
Groundnut (Host)
Buckwheat (Non)
Saltwort (Non)
Bare ground
Groundnut (Host)
Sweet potato (Host)
Soybean (Host)
Sugar beet (Non)
Wheat (Host)
Squash (Host)

17 K. ISOBE ET AL.
content in the soil in agreement with several other studies
[1,2]. In this study, a negative correlation (–0.300) was
also observed between AMF colonization rate and the
amount of available phosphorus in the soil (Table 5).
However, this correlation was not statistically significant.
From this we conclude that the effects of the available
phosphorus in the soil on AMF colonization are smaller
than the effect of the phosphate adsorption coefficient of
the soil on the AMF spore density.
4.2. Genus Glomus Ubiquitous among Soybean
Roots of Kanagawa and Hokkaido
In general, the proportion of the AMF of genus Glomus

K. ISOBE ET AL.
18
Figure 1. Neighbour-joining phylogenetic tree of partial LSU rDNA sequences from AMF colonizing in soybean root collected
from Kanagawa and Hokkaido. KG: Kanagawa, HK:Hokkaido.

K. ISOBE ET AL.
19
Table 2. Soil pH, phosphorus absorption coefficient, available phosphate content and AMF spore density of sampling soil and
AMF colonization rate of soybean roots collected from each sampling site.
Prefecture Site No. pH (H2O) P absorption coefficient Available-P (g/kg)AMF spore density (/g dry soil) AMF colonization rate (%)
Kanagawa
Hokkaido
1
2
3
4
5
6
7
8
9
10
6.6b*
6.4b
7.1a
7.4a
7.2a
6.8ab
6.2b
6.4b
5.4c
5.6c
740d
970c
1480b
1290b
2270a
1830a
460e
1320b
520e
530e
0.28b
0.21bc
0.26b
0.22b
0.07d
0.14c
0.22b
0.42a
0.15c
0.16c
1.7cd
1.8cd
4.2abc
5.4ab
7.0a
6.8a
3.0bcd
3.8bcd
5.1ab
1.3d
16.2bc
5.2ef
1.8f
4.2ef
30.4a
20.1b
12.8cd
18.8b
8.2de
20.5b
*: Means followed by the same letters are not significantly different at 0.05 level according to Tukey’s multiple range test.
Table 3. AMF OTU colonized in soybean root.
Kanagawa Hokkaido
OTU
1 2 3 4 5 6 7 8910
Glo1
2
3
4
5
6
7
8
9
10
11
12
13
Gig1
2
3
Scu1
2
Aca1
2
3
4
Div1
Pra1
Un-known
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
Number of OUT* 11 10 14 89 15 7 475
*: LSU rDNA gene sequences were partitioned into 25 clusters, potentially
yielding 25 OTU (Figure 1). The sequence identity within the clusters
ranged from 97.5% to 100%.
Table 4. t-test of spore number , colonization rate and num-
ber of OTU between Kanagawa and Hokkaido.
Prefecture Spore density
(/g dry soil)
Colonization rate
(%)
Number of AMF
OTU in root
Kanagawa
Hokkaido
4.5 ± 1.0
3.3 ± 0.8
13.0 ± 4.5
15.0 ± 2.8
11.2 ± 1.1
5.8 ± 0.8
Significance ns ns **
ns: no significance between Kanagawa and Hokkaido by t-test. **: 1% level
of significance between Kanagawa and Hokkaido by t-test.
is higher in agroecosystems than natural ecosystems [33-
36]. In the present study, AMF of genus Glomus were
the most abundant colonizer of soybean roots in either
region or site (Table 3). The genus Glomus is known to
be very adaptable to temperature and soil pH [37]. Fu-
thermore, Tarafdar and Praveen-Kumar [38] also consid-
ered Glomus to be the most abundant of all AMF genera
in arid environments. Therefore, Glomus dominates many
soil environments [14,23,37-40]. However, all Glomus
OTUs did not colonize soybean roots equally Glo1 was
found in soybean roots collected from all sites. Glo2,
Glo8, and Glo13 also colonized soybean roots over most
regions and sites (Table 3). We consider that AMF OTUs
Glo1, Glo2, Glo8, and Glo13 are universal to soybean
plants of Kanagawa and Hokkaido. In contract, Glo４
only colonized soybean roots from Kanagawa, and Glo5
colonized soybean roots from Hokkaido (Table 3). We
considered these OTUs to be regionally specific.
4.3. Absence of Gigasporasea in Soybean Roots
from Hokkaido
Lekberg et al. [41] reported that soil type was the one of
most important factors to affect AMF community struc-
ture. But, soils of different types were not dominated by
unique or specific AMF (Table 3). AMF of the family
Gigasporasera were absent from soybean roots collected
from Hokkaido (Table 3, Figure 1). Thus, AMF in the
family Gigasporasea either does not exist or rarely exists
in the soybean fields of Hokkaido. Schenck and Smith
[42] reported root colonization by Glomus ambisporum
was singnificantly greater than that of Gigaspora marga-
rita at 24˚C; however, percent root colonization was
similar for all AMF species at 30˚C. Moreover, spore
production by AMF in the family Gigasporasea was re-
duced under cold temperatures (18˚C) more than for
other families [42]. Hokkaido is a colder region in Japan
and the annual mean temperature is about 10˚C lower
than that of Kanagawa. Thus, colder temperatures many
e responsible for the absence of Gigasporasea in soy- b

K. ISOBE ET AL.
20
Table 5. Coefficient of correlation between AMF spore density or AMF colonization rate and soil pH, phosphate absorption
coefficient, available phosphate content and AMF colonization rate.
pH P absorption coefficient Available-P AMF colonization rate
AMF spore density
AMF colonization rate
0.478
–0.024
0.751*
0.385
–0.421
–0.300
0.242
-
*: 5% level of significance between Kanagawa and Hokkaido by t-test.
bean roots collected from Hokkaido.
4.4. AMF Biodiversity in Soybean Roots between
Regions in Japan
The number of AMF OTUs colonizing soybean roots
collected from Hokkaido was lower (p = 0.01) than that
from Kanagawa (Tables 3, 4). This finding indicates that
the diversity of AMF colonizing soybean roots collected
from Hokkaido is lower than that of Kanagawa. The
community structure of AMF in the soil varies with the
plant species and cropping system [13,43]. And the in-
troduction of AMF non-host crops into a crop rotation
has been shown to decrease AMF biodiversity in soil. In
this experiment, non-host crops and host crops of AMF
were used prior to soybean cultivation in both Kanagawa
and Hokkaido. But, the results of this experiment was
that the number of AMF OTU colonizing soybean roots
collected from Hokkaido was considerably lower than
roots collected from Kanagawa (Tables 3, 4). It should
be concluded that the AMF biodiversity in soybean roots
was more affected by region (Kanagawa or Hokkaido)
than by preceding crop.
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