Effects of soil water stress on nodulation, leaf nitrogenaccumulation and grain yield at three different growth stages ofsoybean
Shakil Uddin AHMED1 , Masateru SENGE2 , Kengo ITO2 and John Tawiah ADOMAKO2

Abstract: An experiment was conducted in a vinyl houseat Gifu University, Japan, from June to November 2008 to
assess the effect of water stress on nodulation of uninoculated soybean and leaf N accumulation to grain yield atthree different growth stages of soybean. The experimental
design was a randomized complete block of five treatmentswith nine replications. The deficit irrigation treatments imposed were D1 (0 – 20 %), D2 (20 – 40 %), D3 (40 – 60 %),D4 (60 – 80 %) and D5 (80 – 100 %) of water deficit total
available water (TAW). The three growth stages were flowering (49 DAS: days after sowing), seed growth (77 DAS),
and maturity (140 DAS). The highest leaf N accumulation
was in the D2 treatment at the flowering and seed growthstage. The soybean grain yield had positive significant correlation (p < 0.01) with leaf nitrogen at seed growth stage.
Total nodule numbers at ≥ 4.75 mm diameter size had nonsignificant effect on leaf N accumulation, but had positive
significant effect (p < 0.05) on grain yield of soybean atseed growth and maturity stage. On the other hand, total
nodule numbers at < 4.75 mm size had positive significant effect (p < 0.01) on leaf N accumulation and grain
yield of soybean at seed growth stage. Total nodule freshand dry weight at ≥ 4.75 mm size had non-significant effect on leaf N accumulation and grain yield of soybean, but
nodules at < 4.75 mm size had a positive significant effect(p < 0.01) at seed growth stage. Individual nodule freshand dry weight at ≥ 4.75 mm size showed negative significant correlation (p < 0.01) with leaf N accumulation andgrain yield, but nodules at < 4.75 mm size showed positive
significant correlation (p < 0.01) at seed growth stage. Ourstudies demonstrated that the water deficit level D2 (20 –40 % of TAW) was the best for an efficient Rhizobium-hostassociation and subsequent nodule development. Based on
our results, it can be concluded that successful root infection of uninoculated soybean was more pronounced in <4.75 mm diameter size class nodule than the larger ones(≥ 4.75 mm) under different water deficit levels.
Key Words : deficit irrigation, leaf N accumulation, nodulation, soybean
1 Graduate School of Agriculture, Gifu University, Yanagido 1-1, Gifu
City, Japan.
2 Faculty of Applied Biological Science, Gifu University, Yanagido 1-1,
Gifu City, Japan. Corresponding author: 千家正照, 岐阜大学応用生物科

学部

2009 年 10 月 14 日受稿 2010 年 1 月 4 日受理
土壌の物理性 114 号, 11–16 (2009)

1. Introduction
Nodulation and leaf nitrogen (N) accumulation in soybean [Glycine max (L.) Merrill] are sensitive to water
deficit conditions, and can have significant effects on yield.
There have been numerous studies on the relationship between soil moisture and activities of soil microorganisms
as well as nodulation (Hill et al., 2000). Soil moisture is
known to affect various physiological processes in plants(Gan et al., 2008). A disturbed water metabolism of themacrosymbiont may cause an impairment of the soil-plantwater balance, which may lead to reduce N2 fixation anduptake (Upreti and Murti, 1999). The soil moisture that is
adequate for seed germination is also adequate for bacterial activity and nodules formation. The soil moisture condition changes with time and may not be sufficient for subsequent nodulation and their potential activities (Ramos etal., 1999). The response of N2 fixation rates to drought isrelated in part to nodule formation and growth (Serraj etal., 1999 ; King and Purcell, 2001).
The ability of legumes to derive N through symbiotic
N2 fixation reduces their dependence on soil N for growth.
However, several factors can affect N2 fixation in legumes.
Kirda et al. (1989) demonstrated that N2 fixation was
the most sensitive parameter to drought, followed by plant
growth, and the least sensitive by soil N uptake. The N2
fixing effectiveness of the legume-Rhizobium symbiosis
has been estimated in various ways. Little is known about
the effect of deficit irrigation scheduling on nodulation andleaf N accumulation at different growth stages of uninoculated soybean.
The present study was, therefore, designed to investigate
the impacts of soil water deficit at different growth stagesof soybean on uninoculated nodulation in two size classes(≥ 4.75 mm and < 4.75 mm diameter), and the subsequentleaf N accumulation in the plant. The factors contributing
to grain yield were also examined.

12

土壌の物理性

Table 1 The soil physical, moisture and chemical properties.
sand: 0.40
Texture (g g−1 )silt: 0.27
clay: 0.33
Physical
Textural classclay loam
properties
Particle density (g cm−3 )2.49
Bulk density (g cm−3 )1.07
Total porosity (m3 m−3 )0.57
Field capacity, θFC (31 kPa) (m3 m−3 ) 0.516
Moisture
Wilting point, θPWP (1553 kPa) (m3 m−3 ) 0.296
properties
Total available moisture, θFC − θPWP
0.220
pH (H2 0)
6.41
Organic matter (g g−1 )
0.065
−1 )
Total
carbon(gg
0.038
Chemical−10.0026
properties Total nitrogen (g g )C/N
13.3
Available phosphorus (g kg−1 )0.164
Exchangeable potassium (mg 100g−1 ) 13.2

2. Materials and methods
2.1 Area description
This research was conducted in a vinyl house (surrounding sides were open) located in the experimental farm ofGifu University (35◦ 27’ N. and 136◦ 44’ E.), Japan, from
June to November 2008. The average temperature was
22.4 ◦ C and the relative humidity was 67.5 % during experiment duration. The soil physical, moisture, and chemical
properties are shown in Table 1.
2.2 Treatments and experimental design
Five water deficit treatments namely; D1 (0 – 20 %), D2(20 – 40 %), D3 (40 – 60 %), D4 (60 – 80 %) and D5 (80– 100 %) water deficit of total available water (TAW) werearranged in a completely randomized block design withnine replications. The water deficit level of D2 (20 – 40%), for example, meant that the available water deficit wasmaintained between 20 % and 40 % of TAW throughout
the growing season. When the maximum allowable depletion of available water came close to 40 % of TAW, water
was applied to restore the available water to the deficit levelof 20 % of TAW. The TAW is defined as the water content
between field capacity (θFC ) and permanent wilting point(θPWP ).
Plastic pots (10 liters volume and 23.8 cm diameter) withno drainage holes were filled with 7 kg air-dried Inceptisol(clay loam in texture). Five soybean seeds [Glycine max(L.) Merrill] were sown in each pot. Prior to planting, water was applied to all the pots to bring them to field capacity (θFC ) for uniform germination. The soil moisture for all
pots was maintained at field capacity (θFC ) until 14 days after sowing (DAS) and the deficit irrigation treatments were
initiated. The growing period of soybean was 20 weeks
from June 16 to November 3. The plants were thinned to
one per pot at the 2 to 3 leaf stage. Three replicate pots ofeach water deficit level were sampled at 49 DAS (flowering

第 114 号

(2010)

stage), 77 DAS (seed growth stage), and 140 DAS (maturity stage) during the experiment. Three pots per treatment
were used for final yield analyses.
2.3 Sampling method
Plants were harvested in a laboratory so that nodule
fresh weights (NFW) could be recorded immediately. Soilwas removed from plant roots, and nodules were separated
from the roots. Nodules were sorted using 4.75-mm wiremesh sieves resulting in two nodule diameter size classes(≥ 4.75 mm and < 4.75 mm). The NFW and the numberof nodules per plant were recorded according to the two
diameter size classes. All plant parts (leaves, stem, root,and nodules by size class) were dried at 65 ◦ C for 96 h anddry weights recorded.
Ground samples of dried soybean leaves were screened
through 1 mm sieve. The leaf N status was determined with
an automatic high sensitive NC analyzer (Sumigraph NC
95 A, Shimadzu Co. Ltd., Japan). Available soil phosphorus for plants was determined by Bray and Kurtz method.

3. Results
3.1 Grain yield and leaf nitrogen accumulation
under different water deficit levels
The grain yield decreased with increasing water deficit
levels (Fig. 1). Significant differences were observed indecreasing grain yield from D3 to D5 , but not in D1 andD2 . The percentage reduction in grain yield compared toD1 , was 1 % for D2 , 12 % for D3 , 21 % for D4 , and 47 %for D5 .
Leaf N accumulation was the highest in D2 treatment,
but decreased up to the D5 at both flowering and seed
growth stages. At the maturity stage, leaf N accumulation increased up to the D3 , and then decreased from D3 to
D5 . Irrespective of the water regime treatment, leaf N accumulation was the highest at the flowering stage and thelowest at the maturity stage (Fig. 2). The soybean grain
yield has positive significant correlation with leaf nitrogenat seed growth stage (Fig. 3).

Fig. 1 The effect of water deficit levels on grain yield of soybean. Means followed by different small letters (a–d) in the column under different water deficit levels are significantly different
according to Tukey’s multiple comparison test (p < 0.05).

論文：Effects of soil water stress on nodulation, leaf nitrogen accumulation and grain yield of soybean

Fig. 2 The effect of water deficit levels on leaf nitrogen at
different growth stages of soybean. Means followed by different small letters (a–d) at each growth stage under different water deficit levels are significantly different according to Tukey’s
multiple comparison test (p < 0.05).

13

Fig. 3 Plot of leaf nitrogen at different growth stages against
grain yield under different water deficit levels. ns: non significant, and ∗∗ significant at p < 0.01.

Table 2 The effect of water stress on nodule development parameters at different growth stages of soybean.

GrowthStage(DAS)

Treatment

D1
D2
49

D3
D4
D5
D1
D2

77

D3
D4
D5
D1
D2

140

D3
D4
D5

TNN
(mg/plant)
Nodule size
class

TNFW
(mg/plant)
Nodule size
class

TNDW
(mg/plant)
Nodule sizeclass

≥ 4.75mm< 4.75mm≥ 4.75mm< 4.75mm≥ 4.75mm< 4.75
mm

①

②

③

④

⑤

⑥

24.0
a
9.0
b
5.0
c
4.0
c
7.3
b
43.7
a
46.0
a
55.0
a
36.3
a
9.3
b
68.7
a
76.7
a
58.7
a
39.0
bc
31.3
c

39.5
a
47.7
a
29.7
b
20.0
bc
12.3
c
48.0
a
55.7
a
42.0
ab
33.3
bc
20.0
c
51.0
a
58.0
a
48.3
a
41.0
b
38.7
b

2800
a
850
b
370
c
500
b
830
b
6000
ab
5610
ab
7980
a
8020
a
2910
c
13010
ab
14310
ab
16700
a
8320
c
6150
c

1580
a
1200
ab
690
ab
490
bc
430
bc
1700
a
1870
a
1470
ab
680
ab
150
c
2310
a
1630
a
940
b
1570
a
2300
a

870
a
260
b
90
c
300
b
270
b
2400
b
2480
ab
3660
a
2980
a
1060
c
3970
a
4020
a
4730
a
2610
b
170
c

530
a
350
ab
220
b
130
c
120
c
670
a
840
a
510
ab
210
b
30
c
700
a
470
b
240
c
510
b
680
a

INFW
(mg/plant)
Nodule size
class

INDW
(mg/plant)
Nodule sizeclass

≥ 4.75< 4.75≥ 4.75< 4.75
mm
mm
mm
mm
⑦=
⑧=
⑨=
⑩=
(③ / ①) (④ / ②) (⑤ / ①) (⑥ / ②)
117
40
36
13
a
a
b
a
94
25
29
7
ab
a
bc
a
74
23
18
7
ab
ab
bc
a
125
25
75
7
a
ab
a
a
113
35
37
10
a
a
b
a
138
36
55
14
bc
a
bc
a
122
34
54
15
bc
a
bc
a
145
35
67
12
b
a
bc
a
221
20
82
6
ab
bc
ab
a
312
8
113
1
a
c
a
b
190
45
58
14
ab
ab
a
a
187
28
52
8
ab
bc
ab
a
285
19
81
5
a
c
a
b
213
38
67
13
ab
bc
a
a
196
59
54
18
ab
a
ab
a

TNN：total nodule number, TNFW: total nodule fresh weight, TNDW: total nodule oven dry weight, INFW: individual nodule fresh weight, INDW:
individual nodule oven dry weight at ≥ 4.75 mm diameter and < 4.75 mm diameter size classes. Means followed by different small letters (a–d) in thesame column in each growth stage under different water deficit levels are significantly different according to Tukey’s multiple comparison test (p <0.05).

14

土壌の物理性

Table 3 Correlation coefficient of nodulation at ≥ 4.75mm and< 4.75mm diameter size class with leaf nitrogen accumulation at
different growth stages of soybean.
Growth stage (DAS)

NoduleNoduleparameter size class

49
77
140
r
p
r
p
r
p
≥ 4.75mm 0.423 ns 0.737 ns
0.002 ns
TNN
＜ 4.75mm 0.868 ns 0.982 0.01 0.011 ns≥ 4.75mm 0.323 ns 0.444 ns
0.398 ns
TNFW
＜ 4.75mm 0.731 ns 0.992 0.01 −0.954 0.05
≥ 4.75mm 0.155 ns 0.553 ns
0.365 ns
TNDW
＜ 4.75mm 0.702 ns 0.981 0.01 −0.918 0.05
≥ 4.75mm −0.556 ns −0.984 0.01 0.677 ns
INFW＜ 4.75mm 0.040 ns 0.960 0.01 −0.810 ns≥ 4.75mm −0.770 ns −0.992 0.01 0.660 ns
INDW＜ 4.75mm 0.106 ns 0.995 0.01 −0.768 ns

第 114 号

(2010)

D2 treatment. The highest fresh and dry weights of nodules ≥ 4.75 mm size were in D4 and D3 treatment, respectively. However, D2 treatment recorded the highest freshand dry weights of nodules < 4.75 mm size. The D5 treatment recorded the highest Individual fresh and dry nodule
weights for nodules ≥ 4.75 mm size. On the other hand,
the highest individual fresh and dry nodule weights at <4.75 mm size were in D1 and D2 treatments, respectively.

r = correlation coefficient, p = probability of significance level, ns =non significant

At maturity stage (140 DAS), the highest nodule number
for both sizes was recorded in D2 treatment. Total and individual nodule fresh and dry weights at larger size ( ≥ 4.75mm) were the highest in D3 treatment. On the other hand,
total nodule fresh and dry weights at smaller size were thehighest in full irrigation treatment (D1 ). Individual nodule
fresh and dry weights at smaller size (< 4.75 mm) were thehighest in sever water stress conditions (D5 ).

Table 4 Correlation coefficient of nodulation at ≥ 4.75mm and< 4.75mm diameter size class with grain yield at different growthstages of soybean.

3.3 Relationship among the nodulation, leaf
N accumulation, and grain yield at different
growth stages

Nodule Noduleparameter size classGrowth stage (DAS)
49

77

140

r
p
r
p
r
p
≥ 4.75mm 0.470 ns
0.891 0.05 0.923 0.05
TNN
＜ 4.75mm 0.917 0.05 0.964 0.01 0.868 ns≥ 4.75mm 0.405 ns
0.558 ns
0.818 ns
TNFW
＜ 4.75mm 0.805 ns
0.964 0.01 −0.296 ns≥ 4.75mm 0.359 ns
0.632 ns
0.867 ns
TNDW
＜ 4.75mm 0.788 ns
0.956 0.01 −0.289 ns≥ 4.75mm −0.303 ns −0.982 0.01 0.047 ns
INFW＜ 4.75mm −0.141 ns
0.964 0.01 −0.661 ns≥ 4.75mm −0.255 ns −0.999 0.01 0.120 ns
INDW＜ 4.75mm 0.013 ns
0.976 0.01 −0.644 ns

r = correlation coefficient, p = probability of significance level, ns =non significant

3.2 Nodulation
The nodule numbers at ≥ 4.75 mm and < 4.75 mm diameter size as well as total and individual fresh and dryweights of nodule are shown in Table 2.
At flowering stage (49 DAS), the highest nodule numberat ≥ 4.75 mm diameter size was recorded in D1 treatment,
while the D2 treatment recorded the highest nodule number at < 4.75 mm diameter. In addition, nodule numberof smaller size (< 4.75 mm) was more than the larger size(≥ 4.75 mm). Fresh and dry nodule weights of both sizes
were higher in full irrigation treatment (D1 ) than the othertreatments. The highest individual nodule fresh and dryweights of larger size class were recorded in D4 treatment,but on the contrary, the highest for the smaller size diameter class in full irrigation treatment (D1 ).
At seed growth stage (77 DAS), the highest nodule number at ≥ 4.75 mm size was recorded in D3 treatment, but
the highest nodule number at < 4.75 mm size was in the

Correlation coefficients of each nodule parameter withleaf N accumulation and grain yield are shown in Table 3
and Table 4.
There was a weak correlation and non-significant relationships among the nodulation, leaf N accumulation, andgrain yield at flowering stage, except total nodule number
at <4.75 mm size that showed significant positive correlation (< 0.05) with yield. Leaf nitrogen and grain yield
showed similar trend of relationships with fresh and dryweights of total and individual nodule weight at both nodule size classes.
On the contrary, at seed growth stage (77 DAS), total
nodule numbers at ≥ 4.75 mm size had non-significant effect on leaf N accumulation, but positive significant effect(p < 0.05) on grain yield of soybean. On the other hand,
total nodule numbers at < 4.75 mm size had positive significant effect (p < 0.01) on leaf N accumulation and grain
yield of soybean. Total nodule fresh and dry weight at ≥4.75 mm size had non-significant effect on leaf N accumulation and grain yield of soybean, but nodules at < 4.75
mm size had a positive significant effect on leaf N accumulation (p < 0.01) and grain yield (p < 0.05). Individual
nodule fresh and dry weight at ≥ 4.75 mm size had negative significant effect, but nodules at < 4.75 mm size had apositive significant effect (p < 0.01) on leaf N accumulation and grain yield at seed growth stage.
The leaf N accumulation and grain yield at maturity
stage (140 DAS) showed non-significant correlation withnodulation parameters, except total nodule fresh and dryweights at < 4.75 mm size showed significant negative
correlation (p < 0.05) with leaf N accumulation, and total nodule number at ≥ 4.75 mm size showed significant
positive correlation (p < 0.05) with grain yield.

論文：Effects of soil water stress on nodulation, leaf nitrogen accumulation and grain yield of soybean

4. Discussion
The highest leaf N accumulation in D2 treatment atflowering and seed growth stages indicated that irrigation
scheduling of 20 – 40 % water deficit of TAW might haveprovided an adequate soil moisture condition that is required for establishing an efficient Rhizobium-host association and subsequent nodule development. This result
agrees with Pahalwan and Tripath (1984) who demonstrated that under uninoculated soybean plant, more leaf N
accumulation were recorded under mild water stress condition. The importance of adequate soil moisture for efficientinteraction of Rhizobium and host was also pointed out by
Gallacher and Sprent (1995).
Our results indicated that water stress conditions did notalways inhibit nodulation but rather sometimes enhance
nodulation. In saturated soil, microbial activity is depressed by poor aeration and the limited availability of O2(Jinfeng et al., 2008). In our full irrigation treatment (D1 ),excessive water might have resulted in poor aeration, andthus reduced the number of aerobic soil microorganisms
as well as nodulation. On the other hand, under mild water
stress conditions (D2 ), facultative anaerobic soil microorganisms might have dominated nodule production. Under
D5 treatment (which is nearer to wilting point), the severe
water stress resulted in an unfavorable growth environment
for the microbes, and this led to the lower nodulation. This
result agrees with Sinclair et al. (1987) that nodulation responds to drought only when the stress was extremely severe, and that the sensitivity was distinctly different fromthe sensitivity of N2 fixation to drought. Clein and Schmel(1994) also found that lower moisture contents inhibitedsoil microbial activity.
Water deficit had significant effect on relationships
among the nodulation, leaf N accumulation, and grain
yield at seed growth stage, because physiological maturity
might reach maximum at that time. Sridhara et al. (1995)
found the same phenomenon that critically important period for fixation and assimilation of nitrogen in soybean
production is during the interval between initial seed formation and the end of the linear seed-filling phase.
Significant positive relationships among the nodulation,leaf N accumulation, and grain yield at < 4.75 mm size
indicates that more successful root infection at < 4.75 mm
size class nodules than the ≥ 4.75 mm size class nodules.

5. Conclusions
Our studies demonstrated that the water deficit levelD2 (20 – 40% of TAW) was the best for an efficient
Rhizobium-host association and subsequent nodule development. Water deficit had significant effect on relation-

15

ships among the nodulation, leaf N accumulation, andgrain yield at seed growth stage, because physiological maturity might reach maximum at that time. Based on our
results, it can be concluded that successful root infectionof uninoculated soybean was more pronounced in < 4.75
mm diameter size class nodule than the larger ones (≥ 4.75mm) under different water deficit levels.
Given the relationship of nodulation and leaf N accumulation with grain yield, it is obvious that no one single
character was important for grain yield. Yield is a complex
terminal outcome of growth to which there are diverse andinterrelated development tracks. However, based on our results, it appears that nodulation, leaf N accumulation, andgrain yield are important characters to consider during soybean cultivation under deficit irrigation practices.

Acknowledgements
The authors are grateful for the financial support of the
Ph.D. program by the University of Dhaka, Bangladeshand supported in part by The United Graduate School of
Agricultural Science, Gifu University, Japan.

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growth of V. faba. J. Exp. Bot., 28: 413–423.
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要

旨水分ストレスがダイズの根粒形成の状況と葉内窒素含有量が収量に及ぼす影響を各生育ステージで調べるために，2008 年 6 月から 11 月にかけて岐阜大学のビニールハウス内で，栽培実験を実施した．実験
方法は 1 因子 5 水準 9 反復の無作為の完全型試験である．すなわち，水分ストレスの処理として，土壌
水分欠損量が総容易有効水分量（TAW）の 0 ∼ 20 % (D1 )，20 ∼ 40 % (D2 )，40 ∼ 60 % (D3 )，60 ∼ 80

% (D4 )，80 ∼ 100 % (D5 ) の 5 水準の処理区を設けた．開花期 (49 DAS)，結実期（77 DAS），成熟期(140 DAS) の 3 段階の生育ステージでサンプリングを行った．葉内窒素含有量は開花期および結実期において D2 試験区で最大となった．ダイズ収穫量は，結実期の葉内窒素含有量と正の有意な相関 (p <0.01) があった．4.75 mm 径以上の根粒の総数は葉内窒素含有量と相関が見られなかったが，結実期，成熟期における根粒数はダイズの収量と正の有意な相関が見られた (p < 0.05)．一方、結実期において4.75 mm 径未満の根粒の総数は，葉内窒素含有量やダイズ収量と正の有意な相関 (p < 0.01) があった．
4.75 mm 径以上の根粒の総乾燥重及び湿潤重は葉内窒素含有量やダイズ収量と相関がなかったが，結実期における 4.75 mm 径未満の根粒の総重量は正の有意な相関があった (p < 0.05)．4 結実期における4.75 mm 径以上の一個体団粒の湿潤重および乾燥重は葉内窒素含有量やダイズ収量と負の有意な相関（ p < 0.01）があったが，4.75 mm 径未満の根粒は正の有意な相関 (p < 0.01) がみられた．以上の結果
から，圃場容水量に対して TAW の 20 ∼ 40 % に相当する水分欠損状態（D2 ）が，根粒菌と宿主の有効な共生関係と根粒の形成にとって最も良好な土壌水分状態であること示している．さらに，結実期における 4.75 mm 径以上の根粒よりも 4.75 mm 径未満の根粒の方が、有効に根粒菌感染していることを示
唆した．
キーワード：節水灌漑，葉内窒素含有量，根粒形成，ダイズ