Field Crops Research 108 (2008) 126–132

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Field Crops Research

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The effects of nitrogen and iron fertilization on growth, yield and fertilizer
use efficiency of soybean in a Mediterranean-type soil
S. Caliskan *, I. Ozkaya, M.E. Caliskan, M. Arslan
Department of Field Crops, Faculty of Agriculture, Mustafa Kemal University, 31040 Antakya, Hatay, Turkey

A R T I C L E I N F O A B S T R A C T

Article history: Poor seed yield of soybean in Mediterranean-type environments may result from insufficient iron (Fe)
Received 20 July 2007 uptake and poor biological nitrogen (N) fixation due to high bicarbonate and pH in soils. This study was
Received in revised form 4 April 2008 conducted to evaluate the effects of N and Fe fertilization on growth and yield of double cropped soybean
Accepted 5 April 2008
(cv. SA 88, MG III) in a Mediterranean-type environment in Turkey during 2003 and 2004. The soil of the
experimental plots was a Vertisol with 176 g CaCO3 kg1 and pH 7.7 and 17 g organic matter kg1 soil.
Keywords:
Soybean seeds were inoculated prior to planting with commercial peat inoculants. N fertilizer rates were
Nitrogen
0, 40, 80, and 120 kg N ha1 of which half was applied before planting and the other half at full blooming
Iron
Early growth
stage (R2). Fe fertilizer rates were 0, 200 and 400 g Fe EDTA (5.5% Fe and 2% EDTA) ha1. It was sprayed as
Agronomic efficiency two equal portions at two trifoliate (V2) and at five trifoliate stages (V5). Plants were sampled at flower
Seed yield initiation (R1), at full pod (R4) and at full seed (R6) stages. Application of starter N increased biomass and
Mediterranean leaf area index at R1 stage whereas Fe fertilization did not affect early growth parameters. N application
continued to have a positive effect on growth parameters at later stages and on seed yield. Fe fertilization
increased growth parameters at R4 and R6 stages, and final seed yield in both years. This study
demonstrated an interactive effect of N and Fe fertilization on growth and yield of soybean in the soil
having high bicarbonate and pH. There was a positive interaction between N and Fe at the N rates up to
80 kg N ha1. However, further increase in N rate produced a negative interaction. Fertilization of
soybean with 80 kg N ha1 and 400 g Fe ha1 resulted in the highest seed yield in both years. We
concluded that application of starter and top dressed N in combination with two split FeEDTA
fertilization can be beneficial to improve early growth and final yield of inoculated soybean in
Mediterranean-type soils.
ß 2008 Elsevier B.V. All rights reserved.

1. Introduction mineralized organic matter, or residual N from the previous crop.

Whereas world soybean production area steadily increased
over the past 20 years production has dramatically decreased in
most Mediterranean countries including Turkey (FAO, 2007). The
decline in production is attributed to unsatisfactory yield, leading
farmers to abandon cultivation. Therefore, identifying major yield-
limiting factors and appropriate agronomic management practices
through field experiments are crucial to increase soybean yield
potential in the region.
Maintaining soil fertility and use of plant nutrients in sufficient
and balanced amounts is one of the key components in increasing
crop yields. Soybean is a legume, and it is capable of biological N2
fixation to produce approximately half of its total N requirement
(Harper, 1987). The rest of N is obtained from soil inorganic N,
* Corresponding author. Tel.: +90 326 245 58 26; fax: +90 326 245 58 32.
E-mail address: scaliskan@mku.edu.tr (S. Caliskan).
This ability has complicated evaluation of the N nutrition of soybean
plants, and conflicting results were often reported in earlier studies
on N fertilization under field conditions. Some researchers
suggested that N fertilization is not necessary for inoculated
soybean (Freeborn et al., 2001; Schmitt et al., 2001; Barker and
Sawyer, 2005; Sogut, 2006) whereas others indicated that biological
N fixation is not adequate to meet the N demand of the crop, and N
fertilization is necessary to improve yield and quality of soybean
depending on application time or rate (Purcell and King, 1996; Gan
et al., 2002, 2003; Osborne and Riedell, 2006; Ray et al., 2006).
Various factors such as temperature, soil type, soil water and organic
matter content, and genotype may affect the response of soybean to
N fertilization. However, there is an agreement in the literature on
the negative effect of large rates of N fertilization on nodulation and
biological N fixation in soybean. Therefore, determination of the
growth and yield response of soybean crop to N fertilization is very
important to maximize yield and economic profitability of soybean
production in a particular environment.

0378-4290/$ – see front matter ß 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.fcr.2008.04.005
 S. Caliskan et al. / Field Crops Research 108 (2008) 126–132
Fe deficiency chlorosis is a widespread problem for soybean
grown on alkaline, calcareous soils (Goos and Johnson, 2000;
Hansen et al., 2003; Heitholt et al., 2003; Wiersma, 2005). Fe
deficiency results in substantial yield loss due to stunted plants
with pale green or yellow interveinal symptoms (Roomizadeh and
Karimian, 1996; Goos and Johnson, 2000; Hansen et al., 2003;
Heitholt et al., 2003; Wiersma, 2005). Fe deficiency also has
negative effect on nitrogenase activity and N fixation by soybean
(Chonkar and Chandel, 1991; Terry and Jolley, 1994). Use of
cultivars resistant to Fe chlorosis is considered as the most
economical approach to overcome deficiency in problem under
chlorosis-prone soils (Goos and Johnson, 2000; Goos and Johnson,
2001; Wiersma, 2005). However, soybean growers may not always
have access to high yielding chlorosis resistant cultivars. Therefore,
much research has been conducted to correct Fe deficiency by
applying various seed, soil or foliar Fe chelates or fertilizers, but
results were contradictory. Goos and Johnson (2000) reported that
foliar application of FeEDTA increased seed yield of soybean, but
seed treatment with FeEDDHA did not. However, the same authors
reported that soil application or seed treatment with FeEDDHA at
high rates can correct chlorosis and increase yield in soybean (Goos
and Johnson, 2001; Goos et al., 2004). Similarly, Wiersma (2005)
suggested that application of FeEDDHA can be effective to reduce
chlorosis and increase yield when applied at high rates. Heithold
et al. (2003) indicated soil application of Fe as FeSO4, FeDTPA or
FeEDDHA did not generate significant and consistent yield increase
in soybean, but they suggested further studies with soil and/or
foliar application of Fe because of the trends for increased seed
yield in some of the Fe treatments. Previous findings clearly
indicate that further studies on soybean response to Fe fertilization
are needed in soybean growing areas prone to Fe chlorosis.
Mediterranean-type soils generally have free CaCO3, high pH,
and low organic matter, and in consequence, Fe deficiency
chlorosis is one of the most important micronutrient disorders
in Mediterranean-type soils (Rashid and Ryan, 2004). Fe status of
the soil affects uptake and use efficiency of macronutrients
(Fageria, 2001; Malakouti, 2008) as well as biological N fixation
process in legume crops (O’Hara, 2001). However, studies on Fe
fertilization of soybean in the Mediterranean basin are sparse.
There are also limited studies on the interaction effect of N and Fe
fertilization on growth and yield of field grown soybean in the
Mediterranean-type environments. This study was aimed to
determine growth and yield response of soybean to N and Fe
fertilization in a Mediterranean-type environment.
2. Materials and methods
Field experiments were conducted at the Experimental Farm
of Mustafa Kemal University in Hatay, located in the Eastern
Mediterranean region of Turkey, in 2003 and 2004. The soil of the
experimental site, developed from alluvial deposits of river
terraces, is typical for the Eastern Mediterranean region of
Turkey, and is classified as Chromoxeret by USDA Soil Taxonomy
(Anon., 1998) and as Vertisol by FAO/UNESCO (1974) having
relatively high clay content with the predominant clay minerals,
smectite and kaolinite. Soil samples were collected from
experimental area at 0–40 cm depth before basal fertilizer
application to test initial soil characteristics, and soil test results
are presented in Table 1. The climate of the experimental site is
typical Mediterranean, with mild and rainy winters, and hot and
dry summers (Table 2).
The indeterminate soybean cultivar SA 88 (maturity group III),
which is widely grown in the region, was used in experiments. The
experimental design was a split plot in randomized complete block
design with three replications. Main plot treatments consisted of
127
Table 1
Initial soil properties of the 0–40 cm soil layer of the experimental site
Soil properties
pHa 7.7
CaCO3 (g kg1) 176.0
Organic matter (g kg1) 17.0
Sand (g kg1) 99.0
Silt (g kg1) 286.0
Clay (g kg1) 615.0
Nb (g kg1) 1.3
Fec (mg kg1) 6.9
Znc (mg kg1) 0.3
Mnc (mg kg1) 3.7
Electrical conductivitya (dS m1) 2.0
Soil order Vertisol
a
pH and electrical conductivity were determined in saturation extracts.
b
N content was determined with Khjeldahl method.
c
Fe, Zn and Mn contents were determined with DTPA extraction method.
four N fertilizer rates: 0, 40, 80, and 120 kg N ha1. Subplot
treatments were three Fe fertilizer rates: 0, 200 and 400 g Fe ha1.
The first half of the N was broadcast by hand and incorporated
immediately before planting. The subsequent part of the N was
side-dressed by hand and incorporated at full flowering (R2) stage.
Urea was used as the N sources in both applications. Fe fertilizer
was applied as two foliar sprays, each consisting of 1.82 kg or
3.64 kg ha1 of FeEDTA (5.5% Fe and 2% EDTA) and 50 ml of
surfactant in 200 l ha1 of water, using a knapsack sprayer. Half of
the Fe doses were applied at the 1–2 trifoliolate stage (V2) and
application was repeated at the 4–5 trifoliolate stage (V5) as
suggested by Goos and Johnson (2000). Plant growth stages were
determined according to methods of Fehr and Caviness (1977).
Seeds were hand planted on 20 June 2003 and 18 June 2004 in six-
row plots, 5 m long with a spacing of 0.7 m between rows, and at a
rate of 25 seeds per meter of row. Seeds were coated with HiStick
soybean inoculant Bradyrhizobium japonicum strain 532C from
Micro Bio Limited (Becker Underwood Inc., USA) to promote N
fixation. Phosphorus (P) was applied at planting to each plot at
80 kg P ha1 as triple super-phosphate. After planting, three light
sprinkler irrigations at 4 days intervals were applied to ensure
uniform germination. The pre-sowing herbicide, trifluralin (a,a,a-
trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine), was applied to soil
at the rate of 960 g a.i. ha1, and the plots were kept weed-free
thereafter by hand weeding. Other cultural inputs were consistent
with local agronomic practices.
Plant samples were taken at 23, 46, and 73 days after planting in
2003, and at 20, 49, and 72 days after planting in 2004;
corresponding to flower initiation (R1), full pod (R4) and full seed
(R6) growth stages, respectively. Ten adjacent plants in the second
row of the each plot were cut at ground level at each sampling, and
plants separated into leaves, petioles, stems and pods. Green leaf
area was measured with a leaf area meter (Model LAI 3100C, LI-
COR, Lincoln, NE), then leaf area index (LAI) was calculated. All
plant samples were dried in an air-forced oven at 70 8C to a
constant weight. Total aboveground dry matter (biomass) was the
sum of all plant parts. Photosynthetic measurements were taken
between 10:00 and 14:00 h on a main stem leaf from the top three
nodes with LCA-4 Portable Photosynthesis System (Model LCA4,
ADC Inc., Herts, UK) at each sampling date. Yield components such
as plant height, number of branches per plant, number of nodes per
plant and number of pods per plant was determined on 20 plants in
each plot before harvest. Seed yield was determined by harvesting
the middle two rows of each plot after adjusting to 130 g kg1
moisture content. Agronomic efficiency (AE; partial derivative of
the function relating economic yield to applied fertilizer nutrient)
and agronomic efficiency for extra fertilizer nutrient increment
128 S. Caliskan et al. / Field Crops Research 108 (2008) 126–132

Table 2
Monthly rainfall, temperature and relative humidity during growing period in 2003 and 2004, and long-term (1992–2004) average

Mean temperature (8C) Rainfall (mm) Relative humidity (%)

Long-term 2003 2004 Long-term 2003 2004 Long-term 2003 2004

June 25.7 25.4 25.6 7.7 10.8 0.0 59.8 67.5 52.5
July 28.6 27.7 28.8 9.6 18.9 0.0 65.1 69.8 45.0
August 27.7 28.6 27.8 6.9 0.0 0.0 66.0 70.2 57.5
September 25.3 25.4 24.9 7.8 8.4 0.0 63.8 62.3 56.5
October 22.6 22.3 22.0 39.0 3.1 3.1 59.8 67.4 47.2

Table 3
Effect of nitrogen and iron fertilization on soybean leaf area index, biomass and photosynthesis rate at three growth stages (R1, R4, R6) in 2003 and 2004

Leaf area index (LAI) Biomass (g m2) Photosynthesis (mmol m2 s1)

2003 2004 2003 2004 2003 2004

R1 R4 R6 R1 R4 R6 R1 R4 R6 R1 R4 R6 R1 R4 R6 R1 R4 R6
1
Nitrogen rates (kg ha )
0 0.76 4.15 5.08 0.87 4.26 4.36 54 283 707 55 370 699 16.6 15.9 12.5 21.3 19.9 17.6
40 0.79 4.69 5.43 0.92 4.58 4.87 55 305 768 60 419 745 16.8 16.6 13.1 21.5 20.1 17.8
80 0.80 5.49 5.49 0.93 4.89 5.20 54 313 756 63 431 748 18.1 16.6 13.7 22.1 20.8 18.1
120 0.81 5.46 5.63 0.94 5.00 5.26 53 308 734 58 426 724 19.0 17.1 13.9 22.6 22.0 18.2

LSD (%5) 0.04 0.40 0.28 0.05 0.22 0.40 2 21 16 4 37 21 0.9 0.4 0.8 0.8 0.7 0.6

Iron rates (g ha1)

0 0.80 4.53 5.13 0.92 4.50 4.93 54 294 719 57 402 686 17.1 16.1 12.7 20.8 20.0 17.7
200 0.79 4.93 5.29 0.90 4.69 4.90 55 301 736 61 416 756 17.5 16.6 13.4 22.3 21.0 18.1
400 0.78 5.39 5.82 0.92 4.86 4.94 55 311 770 60 417 745 18.4 16.8 13.8 22.5 21.0 18.0

LSD (%5) 0.03 0.06 0.08 0.03 0.10 0.20 3 15 22 3 12 26 0.7 0.5 0.5 0.5 0.5 0.5

Nitrogen (N) * ** * ns ** ** ns * ** * * ** ** ** * * ** ns
Iron (Fe) ns ** ** ns ** ns ns ns ** ** * ** ** * ** ** ** ns
N  Fe ** ** ns ns ** ns ns ns ** * ** ns ns ns ** ns ns ns
CV (%) 3.6 4.9 5.3 3.1 2.5 5.1 7.1 5.6 3.4 5.4 3.3 4.1 4.2 3.3 4.0 2.5 2.8 3.1

*, **: F-test significant at P < 0.05, and P < 0.01, respectively. ns: not significant.

(AEex) were calculated for both N and Fe doses according to the
following formulas (Rekha et al., 2005):
pod yield ðfertilizedÞ  pod yield ðcontrolÞ
AE ¼
fertilizer applied
pod yield ðfertilizedÞ  pod yield ðnext lower fertilizer rateÞ
AEex ¼
extra fertilizer increment rate  fertilizer next lower rate
Data for evaluated traits in each year were statistically analyzed
using a standard analysis of variance technique for a split–split plot
design using the general linear model procedure in the Statistical
Analysis System (SAS Institute, 1996). Means were separated using
Fisher’s protected least significance difference (LSD) test at 95%
level of probability.
3. Results
3.1. Leaf area index (LAI)
LAI values were lower than 1.0 at R1 stages, and they rose
sharply until R4 stages in both years. Increase in LAI was small after
R4 stage (Table 3). LAI values increased with N level up to
80 kg N ha1, but further increase in N level did not generate any
significant increase in LAI at R4 stage in both years. Although N
fertilization significantly increased LAI compared to control at R1
and R6 stages, there was no significant difference between N rates
at these growth stages in both years.
Fe application did not show any significant effect on LAI at R1
stages in both years. Noticeable effects of Fe fertilization on LAI were
observed at R4 stage, and LAI values significantly increased with Fe
levels in both years. LAI values increased with Fe levels in 2003
whereas no significant differences were found in 2004 at R6 stage.
Significant N  Fe interactions for LAI at R4 stages were
observed in both years, while the N  Fe interaction for LAI at
R1 stage was found significant only in 2003. No significant
interaction effects between N and Fe was detected for LAI at R6
stages in both years. LAI values increased with increasing N rates
without Fe fertilization whereas LAI values declined in case of large
N (120 kg ha1) application combined with Fe fertilization at R4
stages in both years.
3.2. Biomass
The control plots gave the lowest biomass at all sampling dates
in both years (Table 3). N doses of 40 or 80 kg ha1 produced the
highest biomass at R1 and R6 stages, and further increase in N dose
above 80 kg ha1 resulted in significant decrease in biomass in
both years. However, no significant differences were found among
N doses at R4 stages in both years.
In 2003, biomass values tended to increase with Fe fertilization
at all sampling dates, but significant effect of Fe fertilization was
obtained at R6 stage. Fe fertilization significantly improved
biomass production at all sampling date in 2004. However,
differences between Fe rates of 200 and 400 g ha1 were not
significant in 2004.
3.3. Leaf photosynthesis
Photosynthetic rate showed a decreasing trend from R1 to R6
stage irrespective to N and Fe fertilization in both years (Table 3).
 S. Caliskan et al. / Field Crops Research 108 (2008) 126–132 129

Table 4
Effect of nitrogen and iron fertilization on some yield components and seed yield of soybean in 2003 and 2004

Treatments Plant height (cm) Branches plant1 Nodes plant1 Pods plant1 100-Seed weight (g) Seed yield (kg ha1)

2003 2004 2003 2004 2003 2004 2003 2004 2003 2004 2003 2004

Nitrogen rates (kg ha1)

0 68.1 84.5 3.4 2.5 17.2 16.4 68.2 54.5 15.3 16.6 2069 2179
40 71.4 85.2 3.5 2.7 17.8 17.4 73.2 55.6 15.9 16.9 2345 2209
80 71.8 85.7 3.6 2.9 18.4 18.0 81.8 62.8 15.6 16.2 2505 2468
120 72.2 85.8 3.9 3.0 18.4 17.2 79.0 57.2 15.6 16.4 2673 2410

LSD (%5) 3.1 3.1 0.3 0.1 0.4 0.8 7.3 4.6 0.2 1.5 183.4 73.0

Iron rates (g ha1)

0 68.6 82.7 3.5 2.6 17.8 16.7 73.6 54.7 15.4 16.0 2274 2274
200 70.6 86.0 3.4 2.5 17.9 17.2 76.0 57.5 15.6 16.4 2361 2295
400 73.4 87.1 3.8 2.7 18.2 17.8 76.9 60.4 15.9 17.1 2558 2380

LSD (%5) 1.7 3.8 0.2 0.1 0.4 0.7 3.4 2.5 0.2 0.6 104 25

Nitrogen (N) ns ns * ** ** ** * * ** ns ** **
Iron (Fe) ** * ** ** ns ** ns ** ** ** ** **
N  Fe ns ns ** ns ns ns ** ns ** ns * **
CV (%) 2.8 5.2 7.3 5.3 2.5 4.5 5.2 4.9 1.2 4.3 5.0 1.3

*, **: F-test significant at P < 0.05, and P < 0.01, respectively. ns: not significant.

N  Fe interaction was significant only at R6 stage in 2003. Leaf
photosynthetic rate significantly increased with increasing N
levels from 0 to 120 kg N ha1 at R1 and R4 growth stages in both
years. N fertilization also resulted in higher photosynthetic rate
comparing to control at R6 stages, but differences between N levels
were significant only in 2003.
Fe fertilization significantly increased photosynthetic rate
comparing to control in both years. However increasing of Fe
dose from 200 to 400 g ha1 did not generate noticeable increase in
photosynthetic rate at any sampling, except at R1 stage in 2003.
years (Table 4). The Fe supply significantly improved N utilization of
soybean. Regression curves relating seed yield response of soybean
to N fertilization under different Fe doses were illustrated in Fig. 1.
Seed yield gave almost linear response to N fertilization along with 0
or 200 g ha1 Fe fertilization, and seed yield continuously increased
with N application. However, seed yield of soybean increased until
80 kg N ha1, and further increase in N rate resulted in significant

3.4. Yield components

The effects of N and Fe fertilization on some yield components

and seed yield were presented in Table 4. The numbers of branches,
nodes and pods per plant increased with N doses up to 80 kg ha1,
but further increase in N dose did not show a significant effect on
these traits. The highest 100-seed weight was obtained from
40 kg N ha1 application in both years notwithstanding differences
among N levels were statistically different only in 2003 (Table 4).
Plant height, number of branches per plant and 100-seed
weight increased with increasing Fe doses in both years (Table 4).
The numbers of node and pod per plant also significantly increased
with Fe rates in 2004.
Significant interactions between N  Fe were observed for
number of branch per plant, number of pod per plant, and 100-seed
weight in 2003, whereas no significant effect of N  Fe interaction
were found for any traits in 2004 (Table 4). Although effects of
N  Fe interaction for yield components were not consistent
between years, it was observed that the vegetative growth was
stimulated by N fertilization especially when combined with
400 g Fe ha1 whereas the large amount of N had detrimental
effect on individual seed growth even with the highest Fe supply in
both years. The highest number of branches and nodes were
recorded at the plots of 120 kg N ha1 and 400 g Fe ha1 while the
highest 100-seed weight was obtained from application of
40 kg N ha1 and 400 g Fe ha1 in both years (data were not given).

3.5. Seed yield

The significant main effects of N and Fe fertilization, and Fig. 1. Yield response of soybean to nitrogen rates under different iron rates (values
significant N  Fe interaction for seed yield were obtained in both are means of three replicates).
130 S. Caliskan et al. / Field Crops Research 108 (2008) 126–132

Table 5
Agronomic efficiency of N fertilization under different Fe fertilization rates

Fe rates (g Fe ha1) N rates (kg N ha1) N agronomic efficiency (seed N Agronomic efficiency for extra
yield per kilogram N applied) fertilizer (seed yield per kilogram
extra N applied)

2003 2004 2003 2004

0 40 3.20 0.58 3.20 0.58

80 2.73 2.19 2.25 3.80
120 4.58 1.93 8.28 1.40

200 40 4.88 0.60 4.88 0.60

80 4.80 2.90 4.73 5.20
120 4.94 1.89 5.23 0.13

400 40 12.63 1.08 12.63 1.08

80 8.83 5.73 5.03 10.38
120 5.58 1.96 0.90 5.58

reduction along with high Fe dose (400 g Fe ha1). The highest seed
yields (2794 kg ha1 in 2003 and 2654 kg ha1 in 2004) were
obtained from fertilization with 80 kg N ha1 and 400 g Fe ha1.
Thus, fertilization with 80 kg N ha1 and 400 g Fe ha1 resulted in
seed yield increase around 36 and 22% comparing to unfertilized
control in 2003 and 2004, respectively. However, increase in seed
yield due to N and Fe main effects was only 21 and 13% in 2003 and
13 and 5% in 2004, respectively (Table 4).
N fertilization level are given in Table 6. Although notable
differences in respect to both FeAE and FeAEex values were found
between years, application of 400 g ha1 Fe resulted in higher FeAE
and FeAEex values under each N dose in both years. The highest
FeAE and FeAEex values were obtained from 400 g ha1 Fe
application with 80 kg ha1 N fertilization in both years.
4. Discussion

3.6. Fertilizer use efficiency
Agronomic efficiency of N doses (NAE) and agronomic efficiency
for extra N fertilizer increment (NAEex) under different Fe
fertilization levels are given in Table 5. N and Fe fertilization
considerably increased agronomic efficiency of each other with
combined use. NAE increased with N doses without Fe fertilization
whereas NAE decreased with increasing N doses when combined
with 400 g Fe ha1 in 2003. However, no notable differences for
NAE values were observed between N doses when they applied
along with 200 g Fe ha1 in 2003. NAEx was higher with
120 kg N ha1 under 0 or 200 g Fe ha1 fertilization. The highest
yield increment due to per kilogram extra N fertilization (NAEex)
was obtained from the highest (120 kg ha1) N dose along with 0 or
200 g ha1 Fe fertilization in 2003. With 400 g ha1 Fe fertilization
in 2003, NAEex was the highest with 40 kg ha1 N fertilization, and
it decreased with further increase in N doses. Application of
80 kg ha1 N resulted in higher NAE and NAEex values under each
Fe doses in 2004. Combined use of 80 kg N ha1 along with
400 g Fe ha1 gave the highest NAE and NAEex values in 2004.
Agronomic efficiency of Fe doses (FeAE) and agronomic
efficiency for extra Fe fertilizer increment (FeAEex) under different
The soils used in this study have typical Mediterranean-type
soil characteristics with high CaCO3 and pH level, and low organic
matter content (Table 1). Therefore, carbonate induced Fe
deficiency is expected in the experimental site. This 2-year field
study indicated that soybean crop responded well to both N and Fe
fertilization and combined usage of N and Fe nutrients has
synergistic effect on growth and yield of soybean in this type of
soils.
In calcareous soils with high pH, yield of soybean is often
limited by the lower availability of Fe, especially when they depend
upon symbiosis with root nodule bacteria for their N nutrition. The
present paper reports a comprehensive characterization of the
interactions between N and Fe nutrients in soybean grown in the
soil with high CaCO3 and pH. N and Fe are both involved in vital
plant functions such as photosynthesis, DNA synthesis, protein
formation, respiration and N2 fixation. We found a positive
interaction between N and Fe at the N rates up to 80 kg N ha1.
It is interesting to note that LAI increased with the increasing rate
of N fertilization without Fe, whereas LAI declined with the
combination of higher N rate (120 kg ha1) and Fe in highly
calcareous alkaline soils. Under these conditions soybean had
larger LAI if N fertilizer less than 120 kg ha1 combined with Fe.

Table 6
Agronomic efficiency of Fe fertilization under different N fertilization rates

N rates (g N ha1) Fe rates (kg Fe ha1) Fe agronomic efficiency (seed Fe Agronomic efficiency for extra
yield per kilogram Fe applied) fertilizer (seed yield per
kilogram extra Fe applied)

2003 2004 2003 2004

0 200 0.09 0.04 0.09 0.04

400 0.09 0.07 0.10 0.11

40 200 0.42 0.04 0.42 0.04

400 1.04 0.12 1.65 0.21

80 200 0.92 0.32 0.92 0.32

400 1.31 0.78 1.71 1.24

120 200 0.31 0.02 0.31 0.02

400 0.40 0.08 0.49 0.15
 S. Caliskan et al. / Field Crops Research 108 (2008) 126–132
The comparison of growth responses to N and Fe on calcareous
soils suggests that N utilization of soybean significantly improved
with the supply of Fe. Soybean responded to N fertilization when
combined with no or a moderate level of Fe (0 or 200 g ha1)
fertilization. However, a decrease in seed yield was observed with
the combination of higher N (>80 kg ha1) and Fe (400 g ha1)
rates. The positive interaction indicates that the full response can
be obtained merely by supplying balanced N and Fe nutrients,
since both are yield-limiting factors. On the other hand, further
increase in N rate produced a negative interaction, i.e. Fe addition
replaced the need for increased N. This negative response suggests
that Fe is involved in N fixation, and that supplying Fe also increase
biologically fixed N uptake to the plant, replacing the need for
supply of additional N fertilizer. We found that Fe fertilization
considerably increased agronomic efficiency of fertilizer N, when N
applied at proper dose for not to suppress nodulation and fixation
capacity of Bradyrhizobium bacteria. However, noticeable positive
effect of Fe fertilization on NAE was obtained with higher
(400 g Fe ha1) dose. Fe acts an essential element for mineral
nutrition of rhizobia, nodulation, nodule activity and biological N
fixation (O’Hara, 2001). Previous studies indicated that adequate
Fe supply increased nitrogenase activity and biological N fixation
capacity of soybean (Chonkar and Chandel, 1991; Terry and Jolley,
1994). Apart from the effects of bicarbonate/high pH on decreasing
Fe uptake by the plants, previous studies indicated that
bicarbonate/high pH also impair nodule formation and biological
N fixation by Bradyrhizobium strains in peanut (Tang et al., 1991),
and Lupinus species (Tang and Robson, 1993; Tang et al., 2006).
Tang and Thomson (1996) also reported similar effects of
bicarbonate and high pH on growth and nodulation of a range
of grain legume species. Therefore, a well-balanced N and Fe
fertilization will be necessary to take full advantage of biologically
fixed N uptake.
Although we did not get data on nodulation and biological N
fixation in this study, we can infer from growth and yield results
that nodulation and biological N fixation in soybean was probably
inadequate due to either Fe deficiency or high bicarbonate and pH
in the experimental site. This probably caused insufficient N
nutrition of inoculated soybean without N fertilization. The growth
parameters such as LAI, biomass and leaf photosynthesis
significantly decreased due to unsatisfactory Fe and N uptake.
Establishment of larger leaf area between R1 and R4 growth stages
is very important to improve photosynthetic gain and final yield of
soybean (Kumudini et al., 2001). LAI and biomass values linearly
increased with increasing N rates without Fe fertilization whereas
application of large amount of N had detrimental effect on LAI and
biomass when combined with Fe fertilization. Application of Fe
possibly improved the biological N fixation and reduced to demand
for fertilizer N. On the other hand, N application at high rate might
result in depletion of nodulation and biological N fixation.
However, marked effects of N and Fe rates on growth parameters
such as LAI and biomass were observed at R4 stages. Plants
received only half of total N and Fe doses until the first sampling
date at R1. Lack of Fe response at R1 stages can be attributed to very
short time, only 5 days, interval between the first Fe application
and the first sampling date. Goos and Johnson (2000) also reported
that foliar Fe spray did not reduce chlorosis at sampling 1 week
after spray. Previous studies pointed out that multiple foliar
application of Fe are needed during vegetative growth periods to
correct Fe chlorosis and to improve vegetative growth in soybean
due to short-term effects of foliar Fe supply (Goos and Johnson,
2000; Rashid and Ryan, 2004). Our study indicated that either leaf
area and biomass growth or photosynthetic rate can be increased
with combined usage of N and Fe fertilization under Mediterra-
nean conditions. Increasing photosynthetic rate with N and Fe
131
fertilization can be attributed to increasing amount of chlorophyll
pigments, since N is one of the main components of chlorophyll
and Fe acts in chlorophyll synthesis.
Biological N fixation begins around 2 weeks after planting and
therefore, N uptake from biological N fixation is negligible up to R1
stage (Hardy et al., 1971; Harper and Hageman, 1972). Therefore,
application of a small amount of N at planting called as ‘‘starter N’’
is beneficial to improve early growth and yield of soybean in most
cases (Afza et al., 1987; Starling et al., 1998; Osborne and Riedell,
2006). Our study clearly indicated that application of some starter
N fertilizer improved early growth of soybean plants. However,
differences among N doses were not significant at the earliest
sampling probably due to short duration between emergence and
sampling date (ca. 20 days). We can also deduce from this result
that nodulation and biological N fixation may slow under
experimental condition, which have high bicarbonate and pH.
Previous studies indicated that Bradyrhizobium strains differ
greatly in their ability to nodulation and biological N fixation
under Fe deficiency (Tang et al., 1991; O’Hara et al., 1993). Hence,
we can say that application of starter N at low doses (i.e. between
20 and 40 kg ha1) is enough to stimulate early growth. Higher
amounts of starter N can be harmful for biological N fixation
process. Previous studies pointed out that high amount of starter N
fertilization had detrimental effect on infection, nodulation and N
fixation capacity of Bradyrhizobium bacteria (Beard and Hoover,
1971; Koutroubas et al., 1998). Afza et al. (1987) reported that
lower N rates (less than 40 kg N ha1) did not inhibit N fixation.
Soybean has a relatively high N requirement particularly during
seed filling period, and biological N fixation may not met the crop N
requirement. Therefore, N top dressing during early reproductive
stages can be beneficial to improve growth and yield of soybean. In
this study, we used both starter N fertilization and N top dressing
at R2 stage. Therefore, it is impossible to distinguish the effects of
starter and top dressed N doses at samplings after R1 stage and at
final harvest. Application of 80 kg N ha1 in total
(40 + 40 kg N ha1) resulted in a significant increase in growth
parameters, yield components and seed yield comparing to lower
dose and control. Growth and yield parameters continued to
increase with increasing N doses under zero Fe while they
decreased with increasing N dose over 80 kg ha1 under 400 g ha1
Fe application. This clearly indicates that Fe involved in biological
N fixation. Lack of positive response to the highest starter N rate in
this research can also be attributed to reducing biological fixation
capacity under high N environment. Our results pointed out N
fertilization before sowing and during early reproductive stage can
be beneficial to improve early growth and final yield of inoculated
soybean in case of using proper amount in Mediterranean-type
soils. Similarly, Gan et al. (2003) also reported that application of
starter N at 25 kg ha1 and N top dressing at 50 kg ha1 at early
reproductive stage (R1) increased crop growth and seed yield in
inoculated soybean in China. Our findings are in contrast to
Koutroubas et al. (1998) who reported no significant effect of N
fertilization on growth and yield of inoculated soybean in a
Mediterranean-type environment in Greece. However, Koutroubas
et al. (1998) used very high doses of N (120 and 240 kg N ha1)
without Fe fertilization.
Our results pointed out that some N fertilization can be
beneficial to improve N nutrition of the inoculated soybean, and
Fe fertilization is also required to enhance biological N fixation in
this type of soil. Application of ammonium sulfate can be a better
N source than urea or ammonium nitrate due to its acidifying
properties in this type of soils. Furthermore, B. japonicum strains,
which are tolerant to Fe deficiency, should be selected to
improve soybean adaptation to soils having high bicarbonate
and pH.
132 S. Caliskan et al. / Field Crops Research 108 (2008) 126–132
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