American Journal of Plant Sciences, 2013, 4, 1879-1892
http://dx.doi.org/10.4236/ajps.2013.49231 Published Online September 2013 (http://www.scirp.org/journal/ajps)
Low-Density Co-Inoculation of Myanmar Bradyrhizobium
yuanmingense MAS34 and Streptomyces griseoflavus P4 to
Enhance Symbiosis and Seed Yield in Soybean Varieties
Khin Myat Soe1*, Takeo Yamakawa2
1Plant Nutrition Laboratory, Graduate School of Bioresource and Bioenvironmental Sciences, Faculty of Agriculture, Kyushu Uni-
versity, Fukuoka, Japan; 2Plant Nutrition Laboratroy, Division of Molecular Biosciences, Department of Biosciences & Biotechnol-
ogy, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
Email: *khinmyatsoe@gmail.com
Received June 23rd, 2013; revised July 23rd, 2013; accepted August 15th, 2013
Copyright © 2013 Khin Myat Soe, Takeo Yamakawa. This is an open access article distributed under the Creative Commons Attri-
bution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
ABSTRACT
This study examined whether low-density co-inoculation of Myanmar Bradyrhizobium yuanmingense strain MAS34
and Streptomyces griseoflavus P4 would enhance nodulation, N2 fixation, and seed yield in two soybean varieties. A
field experiment was conducted during the July to November 2012 growing season at Kyushu University Farm, Japan,
using a split-plot design with three replications and the following four treatments: T1, an uninoculated treatment with
peat moss (uninoculated); T2, a single inoculation with S. griseoflavus P4 (P4); T3, a single inoculation of B. yuanmin-
gense MAS34 (MAS34); and T4, a dual inoculation of P4 with MAS34 (P4 + MAS34). Two varieties of soybean,
Yezin-3 (Rj4) and Yezin-6 (non-Rj), were used. The N2 fixation activity of soybean was evaluated by the relative ureide
method using xylem solute from root bled sap at the early pod-fill stage (R3.5). Dry matter production, N2 fixation, and
seed yield were significantly (P < 0.01) different between the inoculated treatments. The effect of variety was also sig-
nificant (P < 0.05) for nodule dry weight at the V6 stage, percentage of N derived from the atmosphere at the R3.5 stage,
and seed yield at the maturity stage. The number of nodules on the tap roots was significantly higher in Yezin-3 than in
Yezin-6. The single inoculation of P4 did not have a significant effect on dry matter production, N2 fixation, and seed
yield in either soybean variety. The dry matter production, relative ureide index, percentage of N derived from the at-
mosphere, and seed yield were significantly (P < 0.01) enhanced by a single inoculation of MAS34 in Yezin-3 and by
dual inoculation of P4 + MAS34 in Yezin-6. These results indicate that low inoculum concentrations (105 cells seed1)
increase N2 fixation and seed yield in these soybean varieties under open field conditions. Myanmar B. yuanmingense
MAS34 and S. griseoflavus P4 are expected to be useful biofertilizers for soybean production.
Keywords: Bradyrhizobium yuan mingense; N2 Fixation; S. Griseoflavus P4; Seed Yield; Soybean
1. Introduction
Soybean (Glycine max L. Merr.) is the most important
grain legume crop in the world and an important protein
source for both humans and livestock [1]. In Myanmar,
soybean has been cultivated for centuries, providing not
only a nutritious food, but also helping to increase the
soil fertility [2]. With demands for this legume increasing
domestically and abroad, soybean has become the second
largest crop cultivated in Myanmar [3].
Soybean helps to maintain soil fertility by assimilating
nitrogen from the atmosphere through symbiotic bio-
logical N2 fixation (BNF) with bradyhizobia [4]. BNF is
of agronomic importance because it reduces the need for
chemical nitrogen fertilizer [5], and nitrogen fixation in
the root nodule is thus an important area of study. The
level of nitrogen fixation varies by cultivar in many le-
gume crop species. Some crops, particularly combina-
tions of strains and cultivars, have been shown to be es-
pecially efficient at N2 fixation [6]. Several studies have
also reported differences between Bradyrhizobium strains
regarding their effectiveness with different soybean ge-
notypes [7-9].
Weaver and Frederick [10] reported that to success-
fully compete with indigenous rhizobium, the introduced
*Corresponding author.
Copyright © 2013 SciRes. AJPS
Low-Density Co-Inoculation of Myanmar Bradyrhizobium yuanmingense MAS34 and Streptomyces griseoflavus
P4 to Enhance Symbiosis and Seed Yield in Soybean Varieties
1880
inoculum must be 1000 times greater. However, in Can-
ada, the standard inoculum for soybean, kidney bean, and
pea is applied at 105 cells·seed1 [11]. Furthermore, Ya-
makawa and Saeki [12] conducted a field experiment at
Kyushu University Farm in Japan to examine the effect
of inoculum density and the use of different inoculation
methods on soybean production. They found that a higher
inoculum density decreased acetylene reduction activity.
Significantly increased yield was found at inoculation
concentrations of 105 cell·seed1 and 107 cell·seed1, but
there was no effect of increasing the inoculum above the
latter density.
Streptomyces griseoflavus P4 was identified by a full
16S rRNA sequencing, and was mostly related to S.
griseoflavus (99.7% identical score) with the GenBank
database accession number of JN102356 [13]. Significant
stimulation effects were observed following the dual in-
oculation of P4 and the Bradyrhizobium strain on N up-
take in adzuki bean, Thai sweet pea [14], and soybean
[15] compared to a bradyrhizobial single inoculation.
Many studies have determined the dry weight and ni-
trogen fixation efficiency of indigenous rhizobial strains
in leguminous plants [16-18]. Our previous laboratory
study revealed that the dual inoculation of S. griseoflavu s
P4 with Myanmar B. yuanmingense MAS34 significantly
influenced the nodule dry weight in Yezin-6 (non-Rj)
soybean and the nodule dry weight and N2 fixation in
Yezin-3 (Rj4) soybean [15]. The experimental data sug-
gested the usefulness of further field investigations of
selected S. griseoflavus P4 and indigenous B. yuanmin-
gense MAS34 with Yezin-3 (Rj4) and Yezin-6 (non-Rj)
soybean varieties. This study was designed to verify the
previous finding and determine whether plant growth and
nitrogen fixation ability were improved in these two soy-
bean varieties under field conditions. Specifically, the
main objective of this study was to evaluate the co-in-
oculation of Myanmar B. yuanmingense MAS34 and S.
griseoflavus P4 to enhance symbiosis and seed yield in
two soybean varieties, using a low density of inoculum
(105 cells·seed1).
2. Materials and Methods
2.1. Experimental Site
The experiment was conducted at Kyushu University
Farm, Fukuoka Prefecture, Japan (33˚37'N, 130˚25'E),
during the July to November 2012 growing season. The
cultivated field had a clay loam soil containing kaolinite
with a pH ranging from 6.1 to 6.4 (soil:water, 1:2.5) or
5.0 to 5.3 (soil:KCl, 1:2.5). The cation exchange capacity
(CEC) of the soil was 20.20 cmolc·kg1 [19]. The soil had
a total nitrogen (N) content of 117 mg per 100 g of soil
[20], total phosphorus (P) content of 76.5 mg per 100 g
of soil [21], available phosphorus (P) content of 9.00 mg
per 100 g of soil [22], and exchangeable potassium (K),
calcium (Ca), and magnesium (Mg) contents of 0.94,
6.31, and 1.69 cmolc·kg1, respectively [19]. To estimate
the density of indigenous rhizobia, soil samples at 15 cm
depth were collected from six locations in the experi-
mental field before fertilization and the rhizobia density
was measured by the most probable number (MPN)
method [23] using two soybean cultivars: Yezin-3 (Rj4-
genotype) and Yezin-6 (non-Rj-genotype).
2.2. Plant Materials
The soybean cultivars Yezin-3 (Rj4) and Yezin-6 (non-Rj)
[24] were collected from the Food Legumes Section, De-
partment of Agricultural Research, Yezin, Myanmar. Rj4
and non-Rj in parentheses indicate the nodulation regu-
latory genes [25]. The Rj genes play a role in controlling
a plant’s compatibility with specific rhizobial strains and
also the preference for indigenous soybean-nodulating
rhizobia [26,27]. In addition, non-Rj is compatible with
all bradyrhizobial strains, but Rj4 has unique features that
restrict nodulation with specific strains of Bradyrhiz o-
bium [28]. Yezin-3 (Rj4) and Yezin-6 (non-Rj) are well-
known soybean cultivars in Myanmar.
2.3. Bacteria Maintenance
The B. yuanmingense MAS34 was stored at 85°C on
HM salts glycerol stock. It was cultured in A1E liquid
media [29] on a rotary shaker (100 rpm) at 30°C for
seven days. The stock culture of S. griseoflavus P4 was
stored at 4°C in IMA-2 medium [30] and cultured in a
broth medium on a rotary shaker (100 rpm) at 30°C for
five days.
2.4. Land Preparation, Inoculation, and Planting
Prior to planting the field was prepared according to con-
ventional practices. Chemical fertilizers containing urea,
super phosphate, and potassium chloride (30.87, 308.75,
and 61.75 kg·ha1) were applied at the rate recommended
by the Department of Agricultural Research, Ministry of
Agriculture and Irrigation, Myanmar. The land was then
leveled and divided into individual plots. The site was 15
m long and 24 m wide. The row width and intra-row
spacing were 60 and 20 cm, respectively.
Peat moss inoculum for 100 soybean seeds was pre-
pared from 1.5 mL deionized water and 10 mL of 12%
aqueous solution of gum Arabic, 10 g of BM2 (raw ma-
terials: peat moss, Group Berger Peat Moss Ltd., Canada),
0.01 mL of 1 × 109 cell·mL1 of B. yuanmingense MAS
34 (1 × 105 cells seed1), and 1 mL of 1 × 107 cell·mL1
of S. griseoflavus P4 (1 × 105 cells·seed1). All inocula-
tion occurred just before planting. Seeds were allowed to
Copyright © 2013 SciRes. AJPS
Low-Density Co-Inoculation of Myanmar Bradyrhizobium yuanmingense MAS34 and Streptomyces griseoflavus
P4 to Enhance Symbiosis and Seed Yield in Soybean Varieties
1881
mix well with the peat moss inoculum prior to seed sow-
ing.
Three inoculated soybean seeds per hill of peat were
sown in the field and immediately covered with rice ash.
A split plot design was used with three replications and
the following four treatments: T1, an uninoculated treat-
ment with peat moss (uninoculated); T2, a single inocu-
lation with S. griseoflavus P4 (P4), T3, a single inocula-
tion of B. yuanmingense MAS34 (MAS34); and T4, a
dual inoculation of P4 with MAS34 (P4 + MAS34). At
the two trifoliate stage (V2 stage), the seedlings were
thinned to two seedlings per hill, and inter-cultivation
and manual weeding were undertaken. Some pesticides
and insecticides were periodically applied when pests
and insects were observed.
2.5. Plant Sampling
Plant samples were collected from three growing stages:
the six unfolded trifoliate leaves stage (V6 stage), early
pod-fill stage (R3.5 stage), and the maturity stage. For
the V6 and R3.5 stages, two plants in one hill per plot
were sampled and separated into nodules, roots, and
shoots. The soil was carefully removed from nodules and
roots by sieving through a 1-mm sieve. Nodules, roots,
and shoots of the plants from each plot were oven-dried
at 70˚C for 72 h for the determination of dry weight. The
shoot dry matter was powdered in a Cyclotec 1093 sam-
ple mill (100 - 120 mesh, Tecator AB, Hoedanaes, Swe-
den). The N content of the dry matter was analyzed using
indophenol [20] after digestion with H2SO4-H2O [31].
For the collection of xylem sap at the R3.5 stage, root
bled sap samples from the plants in each plot were taken.
To collect the root bled sap, the shoot just under the coty-
ledon node of each plant was cut with a very sharp cutter
according to a previously reported method [32]. Sap sam-
ples were kept on ice and frozen at 20˚C for long-term
storage. The root bled sap samples were analyzed for
amino N [33], NO3-N [34], and ureide-N [35]. The rela-
tive ureide index (RUI) of root bled sap at the R3.5 stage
was calculated according to the following formula [32]:


RUI %
4ureide4ureideamino acidnitrate100
The percentage of N derived from N fixation was cal-
culated from y = 21.3 + 0.67x in which y is relative
ureide-N (%) and x is the percentage of N derived from
N fixation (%) [36].
At maturity stage, ten continuous hills were harvested
by cutting at the cotyledon node of the stem. The plants
were harvested from each plot at physiological maturity,
leaving the border rows. The yield components parame-
ters of number of pods per plant, number of seeds per
pod, and hundred-seed weight were determined. After
recording the seed yield, the above-ground dry matters
were oven-dried at 70˚C for 72 h for dry weight deter-
mination. The dry matters were powdered and N content
was analyzed in the same way as for the V6 and R3.5
growth stages.
2.6. Statistical Analysis
Data for the dry weight of nodules, roots and shoots, total
N accumulation, RUI, percentage of N derived from the
atmosphere, and seed yield were statistically analyzed
using STATISTIX 8 (Analytical Software, Tallahassee,
FL, USA) and the means were compared by Tukey’s
HDS test at P < 0.01.
3. Results
3.1. Indigenous Rhizobia in the Cultivated Soil
The density of indigenous rhizobia from cultivated soil
was estimated by the MPN method. Indigenous rhizobia
in this soil nodulated to Yezin-3 (Rj4) and to Yezin-6
(non-Rj) were present at concentrations of 1.16 × 104 and
1.16 × 105 cells (g dry soil)1, respectively.
3.2. Nodulation, Dry Matter Production, and N
Accumulation at the V6 and R3.5 Stages
The number of nodules on tap roots per hill was signifi-
cantly (P < 0.01) different between varieties and treat-
ments (Figure 1). The Yezin-3 variety had a greater
number of nodules on the tap roots than the Yezin-6
soybean variety. A single inoculation of MAS34 resulted
in a significantly (P < 0.01) higher number of nodules on
the tap roots of the Yeizn-3 soybean variety compared to
the uninoculated control at the V6 and R3.5 stages. There
were no significant differences between P4, MAS34, and
the dual inoculation of P4 with MAS34. However, after a
single inoculation of MAS34, 32% and 19% of R3.5-
stage tap roots of the Yezin-3 soybean variety had higher
numbers of nodules than after a single inoculation of P4
and a dual inoculation of P4 with MAS34, respectively.
In the Yezin-6 soybean, the dual inoculation of P4 with
MAS34 was significantly (P < 0.01) higher than the un-
inoculated control in terms of the number of nodules on
the tap roots, but no significant difference was observed
between a single inoculation of P4 and MAS34 at the R
3.5 stage. Nevertheless, following the dual inoculation of
P4 with MAS34, 35% and 17% of tap roots had a higher
number of nodules than after single inoculations of P4
and MAS34 at the R3.5 stage for the Yezin-6 soybean
variety. There was no significant difference in the num-
ber of nodules on lateral roots per hill (Figure 1). How-
ever, a single inoculation of AS34 and the dual inocu- M
Copyright © 2013 SciRes. AJPS
Low-Density Co-Inoculation of Myanmar Bradyrhizobium yuanmingense MAS34 and Streptomyces griseoflavus
P4 to Enhance Symbiosis and Seed Yield in Soybean Varieties
Copyright © 2013 SciRes. AJPS
1882
B
AA
A
a
a
a
a
0
10
20
30
40
50
60
70
80
90
100
UninoculatedP4MAS34 P4+MAS34
Tap rootLateral root
A
AA
A
a
a
a
a
0
20
40
60
80
100
120
140
160
UninoculatedP4MAS34 P4+MAS34
Tap rootLateral root
(a) Yezin-3 at V6 stage
Nodule number per hill
Treatments
(b) Yezin-6 at V6 stage
Nodule number per hill
Treatments
B
AB
A
A
a
a
a
a
0
50
100
150
200
250
300
350
UninoculatedP4MAS34 P4+MAS34
Tap rootLateral root
B
A
A
A
a
a
aa
0
50
100
150
200
250
300
UninoculatedP4MAS34 P4+MAS34
Tap rootLateral root
(c)
Yezin-3 at R3.5 stage
Nodule number per hill
Treatments
(d)
Yezin-6 at R3.5 stage
Nodule number per hill
Treatments
Figure 1. Effects of the co-inoculation of B. yuanmingense MAS34 and S. griseoflavus P4 on nodule number pe r hill at differ-
ent growth stages in the Yezin-3 and Yezin-6 soybean varieties: (a) Yezin-3 at V6, (b) Yezin-6 at V6, (c) Yezin-3 at R3.5, and
(d) Yezin-6 at R3.5. Mean values followed by the same letter(s) indicate no significant difference.
lation of P4 with MAS34 resulted in higher numbers of
nodules on the lateral roots of the Yeizin-3 and Yezin-6
soybean varieties at both stages. Table 1 shows the ef-
fects of the single and dual inoculations of MAS34 and
P4 on nodulation. The efficiency of a single inoculation
of MAS34 was high in tap roots at the V6 stage in Yezin-
3, but no significant effect was found for nodules on the
lateral roots for all treatments at both stages in the Yezin-
3 and Yezin-6 varieties.
The dry weight of nodules was significantly (P < 0.01)
affected by the soybean variety and also the inoculation
treatment at the V6 stage (Table 2). In contrast, it was
not significantly affected by the interaction of the soy-
bean variety and treatment at the V6 stage. All inoculated
treatments had a significantly (P < 0.01) higher nodule
dry weight in both soybean varieties than the uninoculated
control. Yezin-3 had a significantly (P < 0.01) higher no-
dule dry weight (0.41 g·hill1) when inoculated with the
single inoculation of B. yuanmingense MAS34 than did
the Yezin-6 variety (0.22 g·hill1) that received a dual in-
oculation of S. griseoflavus P4 with MAS34.
The dry weight of nodules was significantly (P < 0.01)
improved by the inoculated treatment alone at the R3.5
growth stage (Table 3). In contrast, it was not signifi-
cantly affected by the variety alone or by the interaction
of soybean variety and inoculation treatment at the R3.5
stage. The nodule dry weights following single and dual
inoculations of P4 and MAS34 were significantly (P <
Low-Density Co-Inoculation of Myanmar Bradyrhizobium yuanmingense MAS34 and Streptomyces griseoflavus
P4 to Enhance Symbiosis and Seed Yield in Soybean Varieties
1883
Table 1. Effects of co-inoculation of B. yuanmingense MAS34 and S. griseoflavus P4 on weight per nodule in the Yezin-3 and
Yezin-6 soybean varieties at the V6 and R3.5 stages.
Weight per nodule (mg)
V6 stage R3.5 stage
Treatment
Tap root Lateral root Tap root Lateral root
Yezin-3
Uninoculated 6.27 abc 1.22 a 7.90 a 2.61 a
P4 7.10 ab 1.91 a 7.70 ab 4.14 a
MAS34 8.37 a 1.81 a 5.81 ab 3.60 a
P4 + MAS34 6.74 ab 1.78 a 6.98 ab 3.44 a
Yezin-6
Uninoculated 2.65 c 0.79 a 6.16 ab 2.16 a
P4 3.70 bc 0.97 a 6.43 ab 2.43 a
MAS34 4.12 bc 0.88 a 4.94 ab 2.37 a
P4+MAS34 4.28 bc 0.93 a 4.58 b 2.82 a
Means within a column followed by the same letter(s) indicate no significant difference.
Table 2. Effects of co-inoculation of B. yuanmingense MAS34 and S. griseoflavus P4 on plant dry weight and total N accumu-
lation in the Yezin-3 and Yezin-6 soybean varieties at the V6 stage.
Plant dry weight (g·hill1)
Treatment
Nodule Root Shoot
Total N accumulation
(mg N·hill1)
Varieties
Yezin-3 0.32 a 1.24 a 5.62 a 172 a
Yezin-6 0.18 b 1.10 a 4.00 a 119 a
Treatment of Yezin-3
Uninoculated 0.17 b 0.87 b 3.51 b 108 b
P4 0.33 a 1.31 ab 6.04 ab 186 ab
MAS34 0.41 a 1.38 a 6.01 ab 183 ab
P4 + MAS34 0.40 a 1.41 a 6.96 a 211 a
Treatment of Yezin-6
Uninoculated 0.07 b 0.63 b 2.00 b 56.9 b
P4 0.19 a 1.08 a 3.83 a 116 a
MAS34 0.22 a 1.25 a 4.66 a 141 a
P4 + MAS34 0.20 a 1.43 a 5.23 a 160 a
Tukey HSD test
Variety ** NS NS NS
Treatment ** ** ** **
Variety × Treatment NS NS NS NS
CV% 10.9 12.6 14.7 16.3
N
S, **: nonsignificant or significant at P < 0.01 respectively. Mean values followed by the same letter(s) indicate no significant difference.
Copyright © 2013 SciRes. AJPS
Low-Density Co-Inoculation of Myanmar Bradyrhizobium yuanmingense MAS34 and Streptomyces griseoflavus
P4 to Enhance Symbiosis and Seed Yield in Soybean Varieties
1884
0.01) different in Yezin-3. However, in Yezin-6, the sin-
gle P4 inoculation did not significantly affect nodulation
at the R3.5 stage. The single inoculation of MAS34 pro-
duced a significantly (P < 0.01) higher nodule dry weight
in Yezin-3 but the dual inoculation of P4 + MAS34 did
not produce the same result in Yezin-6.
The dry matter production at the V6 stage was signifi-
cantly (P < 0.01) different between the different inocula-
tion treatments (Table 2). It was not significantly af-
fected by the soybean variety. The Yezin-3 soybean vari-
ety produced a significantly higher amount of dry matter
(1.41 g·hill1 in roots; 6.96 g·hill1 in shoots) following
the dual inoculation of P4 and MAS34. The same re-
sponse was found in Yezin-6 (1.43 g·hill1 in roots; 5.23
g·hill1 in shoots) with the same dual inoculation of P4
and MAS34. The single inoculation of P4 produced a
significant response compared to the uninoculated con-
trol in Yezin-6, but there was no significant effect on dry
matter production in Yezin-3 at the V6 stage. The dual
inoculation of P4 and MAS34 resulted in the highest dry
matter production regardless of soybean variety. How-
ever, the interaction between variety and treatment was
found to be not significant in relation to dry matter pro-
duction.
The dry matter production at the R3.5 growth stage
was significantly (P < 0.01) increased following the in-
oculation treatments (Table 3). However, there was no
significant difference between the soybean variety and
the interaction of variety and treatment. The Yezin-3
soybean variety produced a significantly higher amount
of dry matter following the single inoculation of MAS34.
However, the dual inoculation of P4 with MAS34 sig-
nificantly increased dry matter production in Yezin-6.
The single inoculation of P4 did not significantly affect
dry matter production in Yezin-3 and Yezin-6 compared
with the uninoculated control at the R3.5 stage.
Table 3. Effects of co-inoculation of B. yuanmingense MAS34 and S. griseoflavus P4 on plant dr y weight and total N accumu-
lation in the Yezin-3 and Yezin-6 soybean varieties at the R3.5 stage.
Plant dry weight (g·hill1)
Treatment
Nodule Root Shoot
Total N accumulation
(mg N·hill1)
Varieties
Yezin-3 1.42 a 14.3 a 60.5 a 220 a
Yezin-6 1.36 a 13.5 a 56.0 a 206 a
Treatment of Yezin-3
Uninoculated 0.84 b 13.0 b 50.1 b 196 a
P4 1.51 a 14.5 ab 61.5 ab 211 a
MAS34 1.77 a 15.0 a 65.8 a 238 a
P4 + MAS34 1.52 a 14.9 a 64.3 a 236 a
Treatment of Yezin-6
Uninoculated 0.56 b 11.8 b 48.3 b 189 a
P4 0.94 ab 13.5 ab 53.8 ab 198 a
MAS34 0.83 ab 14.2 a 58.7 ab 211 a
P4+MAS34 1.14 a 14.5 a 63.0 a 224 a
Tukey HSD test
Variety NS NS NS NS
Treatment ** ** ** *
Variety × Treatment NS NS NS NS
CV% 10.9 4.29 7.76 9.07
N
S, *, **: nonsignificant or significant at P < 0.05 or P < 0.01 respectively. Mean values followed by the same letter(s) indicate no significant difference.
Copyright © 2013 SciRes. AJPS
Low-Density Co-Inoculation of Myanmar Bradyrhizobium yuanmingense MAS34 and Streptomyces griseoflavus
P4 to Enhance Symbiosis and Seed Yield in Soybean Varieties
1885
The effects of the co-inoculation of Myanmar B. yuan-
mingense MAS34 and S. griseoflavus P4 in enhancing
the total N accumulation at the V6 and R3.5 stages of the
two soybean varieties are presented in Tables 2 and 3.
The total N accumulation by the soybeans at the V6 stage
was significantly (P < 0.01) different between the inocu-
lated treatments. The effect of variety and the interaction
of variety with treatment had no significant influence on
the total N accumulation by the soybeans at the V6 stage
(Table 2). The dual inoculation of P4 with MAS34 in
Yezin-3 significantly (P < 0.01) influenced the total N
accumulation compared to the other treatments and the
uninoculated control. All inoculated treatments were sig-
nificantly (P < 0.01) better than the uninoculated control
at the V6 stage in Yezin-6.
The total N accumulation at the R3.5 stage was also
observed to be significantly (P < 0.01) affected by the
inoculation treatments. There was no effect of the variety
or of the interaction of variety with treatment on the total
N accumulation by soybeans at the R3.5 stage (Table 3).
There was an insignificant effect on the total N accumu-
lation in Yezin-3 and Yezin-6 compared with the union-
culated control at the R3.5 stage (Table 3). A higher total
N accumulation was observed following the dual inocu-
lation of P4 with MAS34 in Yezin-6 and the single in-
oculation of MAS34 in Yezin-3.
3.3. N2 Fixation at R3.5 Stage
The RUI of root bled sap of soybean at the R3.5 growth
stage [37] was used to indicate the extent of N2 fixation
resulting from the inoculation treatment and the percent-
age of plant N derived from the atmosphere within the
soybean growing season. The RUI and the percentage of
plant N derived from the atmosphere resulting from the
co-inoculation of B. yuanmingense MAS34 and S. gri-
seoflavus P4 in Yezin-3 and Yezin-6 at the R3.5 stage are
presented in Table 4.
Table 4. Effects of co-inoculation of B. yuanmingense MAS34 and S. griseoflavus P4 on N2 fixation in the Yezin-3 and Yezin-6
soybean varieties at the R3.5 stage.
Treatment Relative ureide index (%) Percentage of plant N derived from atmosphere N (%)
Varieties
Yezin-3 67.9 a 69.5 b
Yezin-6 79.2 a 86.5 a
Treatment of Yezin-3
Uninoculated 53.4 b 47.8 b
P4 68.9 ab 71.1 ab
MAS34 77.8 a 84.4 a
P4 + MAS34 71.4 ab 74.8 ab
Treatment of Yezin-6
Uninoculated 74.3 b 79.1 b
P4 74.5 ab 79.4 ab
MAS34 80.3 ab 88.1 ab
P4+MAS34 87.8 a 99.3 a
Tukey HSD test
Variety NS *
Treatment ** **
Variety × Treatment NS NS
CV% 7.98 11.7
N
S, *, **: nonsignificant or significant at P < 0.05 or P < 0.01 respectively. Mean values followed by the same letter(s) indicate no significant difference.
Copyright © 2013 SciRes. AJPS
Low-Density Co-Inoculation of Myanmar Bradyrhizobium yuanmingense MAS34 and Streptomyces griseoflavus
P4 to Enhance Symbiosis and Seed Yield in Soybean Varieties
1886
The RUI and percentage of plant N derived from the
atmosphere were significantly (P < 0.01) different be-
tween the inoculation treatments. They were not signifi-
cantly (P < 0.01) affected by the variety and the interac-
tion of variety and inoculation treatment. In Yezin-3, a
single inoculation of MAS34 (77.8%) significantly (P <
0.01) improved the RUI at the R.5 stage compared to the
uninoculated control (53.2%). The percentage of plant N
derived from the atmosphere from the single inoculation
of MAS34 was about 84.4%. There was no significant
difference between single and dual inoculations of P4 on
the RUI of root bled sap from Yezin-3. The RUI values
of the treatments were within the range of 68.9% - 71.4%,
and plant N derived from N2 fixation ranged between
71.1% and 74.8%. The single inoculation of P4 did not
improve the N2 fixation of soybean, but this treatment
did increase N2 fixation by about 15% over that of the
uninoculated control.
Yezin-6 from the uninoculated control had an RUI
value of about 74.3% for root bled sap at the R3.5 stage.
The single inoculations of P4 and MAS34 had no sig-
nificant effect on the RUI, and they were statistically
equivalent to the uninoculated control. The dual inocula-
tion of P4 with MAS34 (87.8%) resulted in a significant
(P < 0.01) increase in the RUI compared to the other
inoculated treatments and the uninoculated control (74.1%)
in Yezin-6. The percentage of plant N derived from the
atmosphere in the Yezin-6 soybean variety following the
dual inoculation of P4 with MAS34 was about 99.3%
and significantly different (P < 0.01) from the other treat-
ments. The percentages of plant N derived from the at-
mosphere from P4 (79.4%) and MAS34 (88.1%) were
higher than that of the uninoculated control (79.1%).
3.4. Above-Ground Dry Biomass at the Maturity
Stage
The treatment and variety-treatment interaction had a
significant (P < 0.05) effect on the above-ground dry bio-
mass yield. The single inoculation of MAS34 produced a
significantly larger dry biomass yield (80.4 g·hill1) in
Yezin-3 compared to P4 (62.3 g·hill1) and the uninocu-
lated control (41.4 g·hill1). In Yezin-6, the dual inocula-
tion of P4 + MAS 34 significantly increased the dry
biomass (84.7 g·hill1) compared to P4 (66.3 g·hill1) and
the uninoculated control (44.7 g·hill1). In this study, the
variety alone did not produce a significant response in
the above-ground dry biomass yield (Figure 2).
3.5. Total N Accumulation at Maturity
The total nitrogen uptake by soybean plants at maturity
was significantly (P < 0.01) different between the inocu-
lated treatments and the variety-treatment interaction.
The inoculation of B. yuanmingense MAS34 resulted in
the maximum N accumulation (139 kg·N·ha1) at maturity
in Yezin-3, which was significantly higher than with the
inoculation of P4 and in the uninoculated control (Table
4). As in Yezin-6, the dual inoculation of P4 with MAS
34 resulted in a higher N accumulation (148 kg·N·ha1)
at maturity than in the uninoculated control (Figure 3).
c
b
aab
c
bab
a
0
10
20
30
40
50
60
70
80
90
Uninoculated
P4
MAS34
P4+MAS34
Uninoculated
P4
MAS34
P4+MAS34
Incomplete Seed
Complete seed
Unfilled Pod
Shell
St em
Dry matter at Maturity stage (g·hill
1
)
Yezin-3(Rj
4
) Yezin-6 (non-Rj)
Figure 2. Effects of the co-inoculation of B. yuanmingense MAS34 and S. griseoflavus P4 on dry matter at the maturity stage
in the Yezin-3 and Yezin-6 soybean varieties; the four treatments of uninoculated, P4, MAS34, and P4 + MAS34 are shown
on the horizontal axis. Mean values followed by the same letter(s) indicate no significant difference.
Copyright © 2013 SciRes. AJPS
Low-Density Co-Inoculation of Myanmar Bradyrhizobium yuanmingense MAS34 and Streptomyces griseoflavus
P4 to Enhance Symbiosis and Seed Yield in Soybean Varieties
1887
c
bc
a
ab
b
ab ab
a
0
20
40
60
80
100
120
140
160
U ninoculated
P4
MAS3 4
P4+MAS34
U ninoculated
P4
MAS3 4
P4+MAS34
Incomplete Seed
Complete seed
Unfilled Pod
Shell
Stem
Yezin-6 (non-Rj)
N
accumulation at the maturity stage (kg·N·ha
1
)
Yezin-3(
R
j
4
)
Figure 3. Effects of the co-inoculation of B. yuanmingense MAS34 and S. griseoflavus P4 on N accumulation at the maturity
stage in the Yezin-3 and Yezin-6 soybean varieties; the four treatments of uninoculated, P4, MAS34, and P4 + MAS34 are
shown on the The English in this document has been checked by at least two professional editors, both native speakers of
English. For a certificate, please see: http://www.textcheck.com/certificate/rqZzin.
3.6. Yield and Yield-Related Parameters
The number of pods per plant was significantly (P < 0.01)
different among the inoculation treatments in both Yezin-
3 and Yezin-6. The single inoculation of MAS34 resulted
in a significantly higher number of pods per plant than
the single inoculation of P4 and the uninoculated control
in Yezin-3, but it was not statistically different from the
P4 + MAS 34 treatment. In Yezin-6, the dual inoculation
of P4 + MAS34 was significantly different to the union-
culated control. The effect of the soybean variety alone
was not significant in terms of the number of pods per
plant, and the interaction of variety and inoculation treat-
ment also had a non-significant influence on the number
of pods per plant (Table 5).
Although the interaction of variety and treatment did
not significantly influence the number of seeds per pod,
the effect of inoculation treatment and variety alone was
significant (P < 0.01). The single inoculation of MAS34
and the dual inoculation of P4 + MAS34 resulted in a
significantly higher number of seeds per pod than in the
uninoculated control in Yezin-3. In Yezin-6, there was no
significant difference between each treatment (Table 5).
Hundred-seed weight was found to be significantly (P
< 0.01) affected by the effect of inoculation treatment as
well as the interaction between soybean variety and treat-
ment. The single inoculation of MAS34 in Yezin-3 and
the dual inoculation of P4 + MAS34 in Yezin-6 responded
significantly. However, the main effect of soybean varie-
ties alone was non-significant in relation to the hundred-
seed weight (Table 5).
Seed yield varied significantly (P < 0.01) due to the
effects of both soybean variety and inoculation treatment
(Table 5). The higher seed yield in Yezin-3 may be at-
tributed to the larger number of pods per plant recorded
for this variety. The analysis of variance indicated that
variety and treatment had an interaction effect on the
seed yield. The increase in seed yield was significant (P
< 0.01) following the single inoculation of MAS34 (3.45
ton·ha1) in Yezin-3 compared to the uninoculated con-
trol. Significant (P < 0.01) increases in seed yield were
recorded following the dual inoculation of P4 + MAS34
(2.90 ton·ha1) in Yezin-6.
4. Discussion
A successful Rhizobium-legume symbiosis largely de-
pends on the presence of a specific and compatible strain
in the soil for a particular legume. Several studies have
reported a significant increase in soybean growth para-
meters and yield due to the inoculation of bradyrhizobial
isolates [38-40].
Many studies noted that soybean plant nodulation was
inhibited when plants were inoculated with high-density
cell inoculum [12,41-43]. Furthermore, significantly
igher nodulation, fixed nitrogen, and seed yields were h
Copyright © 2013 SciRes. AJPS
Low-Density Co-Inoculation of Myanmar Bradyrhizobium yuanmingense MAS34 and Streptomyces griseoflavus
P4 to Enhance Symbiosis and Seed Yield in Soybean Varieties
1888
Table 5. Effects of co-inoculation of B. yuanmingense MAS34 and S. griseoflavus P4 on seed yield and yield components at the
maturity stage in theYezin-3 and Yezin-6 soybean varieties.
Treatment Pods per hill Seeds per pod Hundred-seed weight (g) Yield (ton·ha 1))
Varieties
Yezin-3 151 a 1.54 a 14.0 a 2.76 a
Yezin-6 165 a 1.11 b 13.0 a 2.02 b
Treatment of Yezin-3
Uninoculated 131 c 1.39 b 12.5 b 1.93 c
P4
MAS34 139 bc
177 a
1.54 ab
1.65 a
14.9 a
14.9 a
2.52 bc
3.45 a
P4 + MAS34 158 ab 1.57 a 14.5 a 3.14 ab
Treatment of Yezin-6
Uninoculated 145 b 1.01 a 11.7 b 1.42 b
P4 151 b 1.13 a 13.2 ab 1.89 ab
MAS34 170 ab 1.07 a 12.4 ab 1.86 ab
P4 + MAS34 191 a 1.24 a 14.5 a 2.90 a
Tukey HSD test
Variety NS ** NS **
Treatment ** ** ** **
Variety × Treatment NS NS * *
CV% 6.74 6.94 4.81 12.9
NS, *, **: nonsignificant or significant at P < 0.05 or P < 0.01 respectively. Mean values followed by the same letter(s) indicate no significant difference; 8.33
hills per square meter.
observed in plants inoculated by low concentrations of
USDA110 under field conditions [12]. In our study, plant
biomass, nitrogen uptake, and seed yield in the Yezin-3
(Rj4) and Yezin-6 (non-Rj) soybean varieties were clearly
enhanced at Kyushu University Farm when they were
inoculated with a low density of (105 cell·seed1) inocu-
lums of B. yuanmingense MAS34 and S. griseoflavus P4.
In this field experiment, soybean plants were grown in
cultivated soil in an open field using ordinary water for
irrigation throughout the experimental period. The plants
from the uninoculated control and the S. griseoflavus P4
treatment were able to form root nodules, indicating that
the indigenous rhizobia in the soil is nodulated to soy-
bean varieties in the open field. This is in line with pre-
vious findings [44] that the response of legumes to in-
oculation depends largely on the number of rhizobia al-
ready established in the soil, the availability of soil N,
and the demand for N by the crop.
The first root nodules formed on the basal part of the
primary roots becoming visible about 10 days after plant-
ing. They started to fix nitrogen (N2) at about 15 - 20
days after planting when the diameter reached approxi-
mately 2 mm [45]. In the later stages, the nodules formed
at the basal part of primary roots degrade, and a large
number of new nodules formed on the lateral roots near
the soil surface. These nodules play an important role in
supplying N during the pod filling stage. In our study, a
number of nodules formed on the tap roots and lateral
roots of soybeans in accordance with the above descrip-
tion and resulted in an improved seed yield.
Souleimanov et al. [46] found that the Nod factor of B.
japonicum had an effect on root growth that resulted in a
34% - 44% longer root in soybean. Significant effects on
root biomass improvement in pea and lentil following
seed treatment with strains of Rhizobium legumanosarum
bv. viceae in field experiments have also been reported
[47]. Studies have also shown results similar to our find-
ing that a single inoculation of B. yuanmingense MAS34
increases root dry weight in Yezin-3 and Yezin-6 soy-
beans at the V6 and R3.5 stages.
In our study, a single inoculation of B. yuanmingense
MAS34 resulted in a significant improvement in N2 fixa-
Copyright © 2013 SciRes. AJPS
Low-Density Co-Inoculation of Myanmar Bradyrhizobium yuanmingense MAS34 and Streptomyces griseoflavus
P4 to Enhance Symbiosis and Seed Yield in Soybean Varieties
1889
tion in Yezin-3 but only a positive trend in Yezin-6. This
supports the conclusions of Wani et al. [48] that within
grain legume species, genotypic variability affects the
nodule number or nitrogenase activity. Comparative as-
sessment of the field trial results suggested that B. yuan-
mingense MAS34 consistently resulted in significant im-
provements in nodulation, dry matter production, N2 fix-
ation, and seed yield in Yezin-3. Increased nodulation
and subsequent nitrogen fixation resulted in increased
plant growth and grain yield. Similarly, Egamberdiyeva
et al. [49] noticed a positive effect of inoculation with B.
japonicum S2492 on growth, nodule number, and yield
of soybean under field conditions.
Milic et al. [50] also reported variability in the dry
matter mass and nitrogen content in the nodules of soy-
bean varieties following the use of different bradyrhizo-
bial strains. Such variations may be attributed to differ-
ences in the genomic constitution of the host or bacteria
or both, which control symbiosis, or there might be more
than one affinity group within the legume rhizobia. Ac-
cording to Okereke et al. [7] the inoculation response of
Bradyrhizobium in different soybean cultivars is cultivar-
and site-specific. We also observed variations between
the two tested soybean varieties in terms of their response
to bradyrhizobial strain.
In both soybean varieties, S. griseoflavu s P4 had a sig-
nificant effect only on the improvement of plant dry
weight at the V6 stage and dry matter production at ma-
turity was significantly better than in the uninoculated
control. Soe et al. [15] stated that S. griseoflavus P4
alone could improve the shoot dry weight of Myanmar
soybean varieties. In the present study, the co-inoculation
of MAS34 with P4 significantly improved nodule dry
weight, plant biomass, N2 fixation, and seed yield in
Yezin-6 but not in Yezin-3. This supports the results of
our previous study [51], in which the dual inoculation of
endophytic actinomycetes (Streptomeces sp. strain, P4)
with bradyrhizobial strains increased the nodulation and
nitrogen fixation in some soybean varieties but not in
other varieties.
Rj genes play a role in controlling the plant’s compati-
bility with specific rhizobial strains and also the prefer-
ence for indigenous soybean-nodulating rhizobia [26,27].
In addition, non-Rj is compatible with all bradyrhizobial
strains but Rj4 has unique features that restrict nodulation
with specific strains of Bradyrhizobium [28]. In this study,
Yezin-3 (Rj4) responded significantly to the single inocu-
lation of B. yua nmingen se MAS34. The MAS34 was iso-
lated from a Rj4-genotype host [15]. These findings sup-
port the results of previous studies [26-28]. The Rj-geno-
type of soybeans appears to be compatible with the pref-
erence of bradyrhizobial strains for soybean cultivation.
Inoculation with effective strains of bradyrhizobia may
promote soybean growth and seed yield. We found that
the symbiotic interaction of the P4 with B. yuanmingense
MAS34 significantly improved nodule dry weight, ni-
trogen fixation, and seed yield in Yezin-6. This synergis-
tic efficacy of S. griseoflavus P4 with B. yuanmingense
MAS34 was found in Yezin-6 (non-Rj). This also sup-
ports previous findings showing that the dual inoculation
of bradyrhizobial strains and S. griseoflavu s P4 increased
nodulation and nitrogen fixation in different soybean va-
rieties [51,52].
Synergistic effects of the co-inoculation of S. griseofla-
vus P4 with B. yuanming ense MAS34 have been found in
Yezin-6 grown at Kyushu University Farm, Japan. This
positive interaction was observed under standard envi-
ronmental conditions, using the correct varieties, and pro-
per nodulated bacteria in conjunction with S. griseoflavus
P4. Our experimental results confirm the findings of Aka-
rapisan et al. [52] and Soe et al. [51] that S. griseoflavus
P4 is an effective endophytic actinomycete, which can be
used in combination with selective root nodule bacterial
strains for the production of economically important le-
guminous crops. Further experimental investigations of
the synergistic effectiveness of selected B. yuanming ense
MAS34 and S. griseoflavus P4 with different soybean
cultivars are needed to determine the optimal Myanmar
soybean growing environment where this MAS34 was
isolated. We envisage that the Myanmar Bradyrhizobium
strain and S. griseoflavus P4 will be useful as biofertiliz-
ers for soybean production in the future.
5. Acknowledgements
This work was supported by grants from the Ministry of
Education, Culture, Supports, Sciences and Technology
of Japan.
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Low-Density Co-Inoculation of Myanmar Bradyrhizobium yuanmingense MAS34 and Streptomyces griseoflavus
P4 to Enhance Symbiosis and Seed Yield in Soybean Varieties
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