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Published May 5, 2015

Crop Economics, Production & Management

Soybean Response to Single or Mixed Soil


Amendments in Kashmir, Pakistan
Abdul Khaliq and M. Kaleem Abbasi*
ABSTRACT

The application of animal- and plant-derived organic substrates with minimal additions of commercial N fertilizers is an important
management strategy for sustainable agriculture production systems in mountain upland soils subjected to continuous erosion. A 3-yr
(2009, 2010, and 2011) field experiment was conducted to evaluate the effects of separate and combined use of poultry manure (PM),
wheat (Triticum aestivum L.) milling residues (WMR), and urea N (UN) on the productivity and N2 fixation of rainfed soybean [Glycine
max (L.) Merr.] grown in the Himalayan region of Rawalakot Azad Jammu and Kashmir, Pakistan. The experiment was conducted in a
randomized complete block design with three replications. Treatments included UN100, PM100, WMR100, PM50 + WMR50, UN50 +
PM50, UN50 + WMR50, UN50 + PM25 + WMR 25, and an unfertilized control. Nitrogen from all amendments was applied at an
equivalent rate of 100 kg total N ha1. Compared with the control and UN100 treatments, the organic amendments applied alone or
combined with UN significantly increased the number and mass of root nodules and N2 fixation by 36 to 68, 26 to 62, and 8 to 31%,
respectively. Poultry manure (PM100) displayed the highest nodulation (49 and 129 nodules) and N2 fixation (43 and 73 kg N ha1),
while UN100 did not differ from the control. Yield and yield components (residue dry matter yield and seed yield) and N use efficiency
in the combined treatment UN50 + PM50 was comparable to that recorded under the UN100 treatment. There was a positive correlation
between nodule number and mass with N2 fixation (r = 0.86 and 0.79), suggesting that root nodulation can be used as an indicator for
N2 fixation. Results of this study confirmed the beneficial effects of organic amendments on N2 fixation and in combination with UN
represents a successful and sustainable management strategy for soybean production in mountainous ecosystems.

Organic inputs of plant and animal origin, such as crop resi-

Supplemental material available online. Dep. of Soil and Environmental


Sciences, Faculty of Agriculture, The Univ. of Poonch, Rawalakot, Azad
Jammu and Kashmir, Pakistan. Received 12 July 2014. Accepted 4 Jan. 2015.
*Corresponding author (kaleemabbasi@yahoo.com).

the overall physical and microbiological environment of the soils


(Barzegar et al., 2002; Zingore et al., 2008).
Soybean is generally considered a high-N-demand crop, and it has
been reported that approximately 300 kg N is needed to produce 3
Mg ha1 of soybean, whereas rice (Oryza sativa L.) requires only 100
kg N to produce 5 Mg ha1 (Gan et al., 2003). Under field conditions, effectively nodulated soybean may obtain >80% of its total N
from N2 fixation (Hungria et al., 2006). However, values around
60% N derived from the atmosphere (Ndfa) are more typical for
temperate conditions (Peoples et al., 1995). Most of the results on
soybean N2 fixation are from conventionally grown soybean, and
there is little information on N2 fixation under organic cropping
systems. Oberson et al. (2007) estimated symbiotic N2 fixation
of soybean under organic and conventional cropping systems and
reported that N2 fixation was lower in conventionally than organically grown soybean. Quantification of N2 fixation by soybean was
examined in India, and the results indicated a significant reduction
in %Ndfa at each increased level of fertilizer N, whereas farmyard
manure (FYM) and integrated treatments displayed progressive
increases in N2 fixation (Singh et al., 2004). The organic matter
buildup in soil due to the addition of organic amendments improved
soil physical conditions and enhanced microbial activity that

Published in Agron. J. 107:887895 (2015)


doi:10.2134/agronj14.0359
Copyright 2015 by the American Society of Agronomy, 5585 Guilford
Road, Madison, WI 53711. All rights reserved. No part of this periodical
may be reproduced or transmitted in any form or by any means, electronic or
mechanical, including photocopying, recording, or any information storage and
retrieval system, without permission in writing from the publisher.

Abbreviations: DMY, dry matter yield; FYM, farmyard manure; Ndfa,


nitrogen derived from the atmosphere; NAE, agronomic efficiency of applied
nitrogen; NPE, physiological efficiency of applied nitrogen; NUE, nitrogen
use efficiency; PM, poultry manure; TSW, thousand-seed weight; UN, urea
nitrogen; WMR, wheat milling residues.

dues, cattle manure, poultry manure, waste materials, and their


composted products are available in abundance in many parts
of the world and their use for improving soil fertility and plant
productivity is an important management strategy for sustainable
agriculture production systems, especially in subhumid, rainfed
soils low in organic matter (Kumar and Goh, 2003). Concerted
efforts have been made to use these organic materials as sources of
major plant nutrients. This possibility has been practiced in many
agroecosystems because increased recycling of organic substrates is
likely to release a substantial amount of N into the mineral N pool,
reducing fertilization costs and providing an amount of available N
that allows maintenance of crop yields (Panwar et al., 2010; Ribeiro
et al., 2010; Azeez and Van Averbeke, 2010; Abbasi and Khizar,
2012). The addition of these organic substrates can effectively restore
and improve the productive potential of soils by supplying multiple
nutrients, increasing soil organic matter content, and improving

A g ro n o my J o u r n a l Vo l u m e 107, I s s u e 3 2 015

887

eventually increased nodulation and N2 fixation (Singh et al., 2004;


Singh and Shivakumar, 2010).
Previous studies have indicated that application of N through
organic amendments including poultry manure increased soybean
grain yields, growth characteristics, N and P uptake (Ramesh et al.,
2009; Bandyopadhyay et al., 2010), and root nodulation (Ghosh et
al., 2004; Tagoe et al., 2008; Chiezey and Odunze, 2009). Results
from a field experiment conducted on a Vertisol in India indicated
that the average seed yield of soybean in an NPK + FYM treatment
was 1830 kg ha1, i.e., 16 and 103% higher than seed yield in the
NPK alone and control treatments, respectively (Hati et al., 2006).
The effect of sole application of inorganic fertilizers (NPK) and
combined application of NPK + FYM on soybean was studied in a
deep Vertisol in India where NPK + FYM significantly increased
seed yield by 14% over NPK alone and by 50% over the control
treatment (Bandyopadhyay et al., 2010). Similarly, incorporation of
plant or crop residues into the soils improved crop yield by providing
N for an extended period of time and therefore reducing N losses in
a soilplant system (Chaudhary et al., 2014).
Combined application of available organic sources along with
an optimal dose of inorganic fertilizers assures high and sustained
productivity in a cereallegume cropping system due to regulated
nutrient supply and reduced losses, besides lowering costs (Manna
et al., 2005). Keeping in view the possible beneficial effects of mixed
organic and inorganic amendments on soybean yield and its effect
on N2 fixation, the present study was planned to assess the effects of
poultry manure and wheat milling residues applied alone or combined with urea N on the growth, yield, nutrient uptake, nodulation,
and N2 fixation of soybean in the Himalayan region of Kashmir,
Pakistan. The results and findings reported here have specific implications for using organic and inorganic amendments to improve
soybean productivity in many other areas and countries.

MATERIALS AND METHODS


Sites Description
The field experiment was initiated during the Kharif (summer, JuneOctober) 2009 season and was performed for three
consecutive years, 2009 to 2011, at two locations: (i) Rawalakot at
the research farm of the Faculty of Agriculture, The University of
Poonch; and (ii) Hajira in a farmers field. The field site at Rawalakot
is located at 335132.18 N, 734534.93 E and an elevation of
1638 m asl, and the field at Hajira is located at 334618.12 N,
735345.96 E and an altitude of 966 m asl. Both sites are located
in the northeast of Pakistan under the foothills of the Great
Himalayas district Poonch, Azad Jammu and Kashmir. The soils
at the study sites were loam in texture, classified as Humic Lithic
Eutrudepts (Inceptisols). The climate of the region is subtemperate. Mean daily maximum and minimum air temperatures at
the Rawalakot site ranged from 27 to 29C (JuneJuly) and 1.0
to 3.5C (JanuaryFebruary). The maximum and minimum
temperatures at the Hajira site was relatively higher, and the corresponding values for the study years were a maximum of 38 to 41C
(JuneJuly) and a minimum of 2.6 to 1.7C (JanuaryFebruary).
The mean (average of 3 yr) annual rainfall was 1388 mm, with 45%
of the total precipitation during June to September and 43% during
January to April. The monthly precipitation and temperature of
the experimental areas during the growing season are presented in
Supplemental Table 1.
888

Experimental Procedures and Details


Before the onset of the experiment, soil samples from 0 to 15 and
15 to 30 cm were collected from both locations, air dried, ground
to pass a 2-mm sieve, and stored in sealed plastic jars before analysis.
The physical and chemical properties of the soil used in the study
are presented in Supplemental Table 2. Soil pH was determined in
a 1:2.5 (w/v) soil/water suspension. Soil organic C was determined
by oxidizing organic matter in the soil samples with K2Cr2O7 in
concentrated H2SO4 followed by titration with ferrous ammonium
sulfate (Nelson and Sommers, 1982). Total N was determined by
Kjeldahl digestion, distillation, and titration (Bremner and Mulvaney,
1982). Extractable P was determined according to Ryan et al. (2001)
using the NH4HCO3diethylenetriaminepentaacetic acid (DTPA)
method modified by Soltanpour and Workman (1979). Exchangeable
K was determined using a flame photometer following soil extraction
with 1 mol L1 NH4OAc (Simard, 1993). The NH4HCO3DTPA
extraction procedure was used for micronutrients, i.e., Fe, Mn, Zn,
and Cu, using atomic absorption spectrophotometry (Soltanpour and
Workman, 1979). Bulk density was determined from undisturbed soil
cores taken from the upper horizon (015 cm) at about five locations
in each field (Blake and Hartge, 1986).
Poultry manure (PM) was collected from a local poultry farm
located at Trar Rawalakot. Wheat milling residues (WMR) were
collected from a wheat flour mill located near Rawalakot, at Arjah
(district Bagh), Azad Jammu and Kashmir. A composite sample of
well decomposed PM was air dried, crushed into smaller particles by
hand pressing, and passed through a 2-mm sieve. Similarly, WMR
were dried in an oven at 65 to 70C overnight, milled, and sieved
through a 1-mm sieve. Samples of both organic materials were stored
in tightly closed plastic containers before chemical composition analysis. Triplicate samples from each of PM and WMR were analyzed
for chemical constituents (Supplemental Table 3). Total N in the
PM and WMR was determined by the Kjeldahl method described
above. Organic matter content was estimated from the weight loss on
ignition at 400C (Ball, 1964). Wet combustion in a 2:1 mixture of
HNO3 and HClO4 was used for before mineral element analysis of
the samples. The P content was determined spectrophotometrically
by the vanadomolybdate yellow method, and the K content was
determined by flame photometer. The lignin content was determined
using the methods of Van Soest et al. (1991). Soluble polyphenols were
extracted in hot water (100C, 1 h) and determined by colorimeter
using FolinDenis reagent (Folin and Denis, 1915).
For proper seedbed preparation, fields at both the sites were tilled
two to three times to a depth of about 20 cm with a simple cultivator attached to a tractor, followed by smoothing with a wooden
planker. However, in subsequent years of the experiment, fields were
plowed (tilled) manually within the studied plots in the respective
plots, 3-m long and 2-m wide, to avoid mixing the soils outside the
plots. Amendments were applied to the same plots for a total of six
cropping cycles.
Treatments and Experimental Design
The treatments were comprised of a control (no N) and all combinations of three sources of N, i.e., PM, WMR, and mineral N as
urea N (UN). Equivalent rates of total N were applied in each treatment. The treatments included: a control without any amendment
(unfertilized control); 100% poultry manure (PM100); 100% wheat
milling residues (WMR100); 100% urea N (UN100); 50% PM and
50% WMR (PM50 + WMR50); 50% UN and 50% PM (UN50 +

Agronomy Journal Volume 107, Issue 3 2015

PM50); 50% UN and 50% WMR (UN50 + WMR50); and 50%


UN, 25% PM, and 25% WMR (UN50 + PM25 + WMR 25). The
experiment was arranged as a randomized complete block design
with three replications.
Well-decomposed PM and dried WMR were pulverized and
then broadcast and incorporated into the soil at the time of sowing.
However, UN was applied in three splits, with one half broadcast by
hand immediately before sowing and the remaining amount split into
two equal doses and sidedressed manually at the R2 (full flowering)
and R4 (full pod development) stages. In integrated N treatments
where UN was applied with PM or WMR, the UN was also applied
in three splits. Basal doses of P and K were applied at the rate of
50 kg ha1 P and K (each) to all plots at sowing, including the control,
in the form of single superphosphate and sulfate of potash, respectively. These treatments were applied to the same plots each of the 3 yr.
Seed of soybean cultivar NARC-1 was initially collected from the
seed and oil section of the National Agricultural Research Centre
(NARC), Islamabad, Pakistan. For the next 2 yr, the harvested
seeds were well mixed before planting and thereafter used in the
experiments. Sowing of soybean was completed in the last week of
May each year. Soybean was planted at a seeding rate of about 80 to
100 kg seed ha1. The seeds were sown by hand to a depth of about
5 cm. A row-to-row distance was maintained at 67 cm. After germination, the soybean plant density was adjusted to about 330,000
plants ha1 by removing the smaller, weaker, and diseased plants.
The resulting plants were reasonably well distributed within the plots
without affecting the population density. All standard local cultural
practices were followed when required throughout the growth period.
No irrigation was provided, and manual weeding was performed on
three occasions during each season.

Measurements
A set of three plants from each plot was randomly selected at full
flowering (the R2 stage) for determining root nodulation (number and mass of nodules) and shoot and root characteristics. The
selected plants were uprooted with a ball of soil for observations
on nodulation. Keeping the root portion intact, the ball of soil was
washed gently with clean running water followed by washing with
camel-hair brushes to dislodge soil particles. Nodules were removed
from the roots, counted, and the dry mass was measured (Vincent,
1970). Shoot length was measured from the base of the plant to the
tip of the final leaf with the use of a meter rod. Leaf area was measured by a CI-203 portable laser leaf area analyzer (CID Biosciences
Inc.) on five freshly taken turgid soybean leaves. Chlorophyll content was measured following the method of Bansal et al. (1999), as
reported by Amujoyegbe et al. (2007). For this purpose, 100 mg of
fresh leaves was taken (at the V8 stage), crushed in 20 mL of 80%
acetone, and the extract was centrifuged for 10 min at 1000 rpm.
Absorbance of the supernatant was recorded at 645 and 663 nm in a
T-80 spectrophotometer. Chlorophyll content (expressed as mg g1
of each sample) was estimated according to Bansal et al. (1999).
At physiological maturity (R8), plants from the three interior
rows in each plot were harvested, tied into bundles, and then left in
their respective plots for drying for about a week. To avoid leaf loss
or pod shattering during drying and handling, the weight of the
bundles (biomass yield) was recorded in the field. The dry matter
yield (DMY) was calculated on a dry mass basis from the difference
in weights of the soybean bundles and seed yield. Soybean seeds

were collected by hand threshing the harvested bundles, and the


seed yield was adjusted to a moisture content of 130 g kg1.

Biochemical Analysis of Plant and Seed Samples


Before harvesting, five soybean plants from each plot were sampled
(at physiological maturity) by cutting the plants at ground level. The
plant samples were dried in an oven at 65C until constant weight.
The samples were ground in a Wiley mill (Polymix PX-MFC 90D)
to pass through a 1-mm sieve. Total N in the aboveground material
was estimated by the Kjeldahl methods described above. Total P and
K were determined by digesting 0.25 g of material with H2SO4 and
H2O2. The P in the digests was measured by spectrophotometer
(Murphy and Riley, 1962), and K was determined by atomic absorption spectrophotometer (Winkleman et al., 1990). Seed samples were
dried at 65C for about 48 h, ground to pass through a 1-mm sieve,
and then analyzed for N, P, and K concentrations using the procedures described for plant analysis. The NPK uptake was calculated
from the DMY and NPK concentration in the respective plant parts
(Fernandez et al., 2008; Abbasi and Tahir, 2012). Total NPK uptake
was the sum of straw and seed NPK uptake.

Estimation of Dinitrogen Fixation


Dinitrogen fixation was assessed by the xylem-solute technique
after collecting sap from the plants at the pod-filling stage (R4) by the
vacuum extraction method (Peoples et al., 1989; Hayat et al., 2008).
In this procedure, the concentration of ureides from petiole-tissue
extracts is expressed as the relative abundance of ureides, which is the
fraction of the N from ureides relative to the sum of the N found in
ureide and NO3 concentrations (Purcell et al., 2004). The concentrations of ureide-, NO3, and amino-N were determined and used to
calculate the relative abundance of ureide N (RUN) and %Ndfa (proportion of plant N derived from N2 fixation) as:
RUN(%) =

4 (ureide conc.)
100
4 (ureide + nitrate + amino N)

where the ureide concentration is multiplied by four because there


are four N atoms in ureide molecules. After getting the value of
RUN, the %Ndfa was estimated as:
%Ndfa = 1.6(RUN-15.9) for plants during R 4

The amount of N2 fixed (at R4) by a legume can be regulated by two


factors, the amount of N accumulated during growth and the production of N derived from symbiotic N2 fixation:
Amount of N2 fixed at R 4 ( kg ha -1 ) =
%Ndfa crop N( kg ha -1 )1.5

The 1.5 factor was used to include an estimate for the contribution
by belowground N (Peoples et al., 1989).

Nitrogen Use Efficiency and its Components


The N contents and N uptake in both the seed and straw of soybean were used for calculating the different N efficiency parameters,
i.e., the agronomic efficiency of applied N (NAE), the physiological
efficiency of applied N (NPE), and the N use efficiency (NUE),
by the methods described by Nyiraneza and Snapp (2007) and

Agronomy Journal Volume 107, Issue 3 2015

889

reported by Abbasi and Tahir (2012). To consider N from N2 fixation, the fixed N (kg ha1) was subtracted from the total plant N
(total plant N uptake), and the NAE, NPE, and NUE calculations
were made with the remainder.

Statistical Analysis
Analyses of variance (ANOVA) were performed using the
MSTAT-C statistical analysis package (Michigan State University) to
determine treatment effects on growth and soybean yield components
in addition to nodulation and N2 fixation at R4. All statistical comparisons were made at the P = 0.05 probability level unless otherwise
stated, using the least significant difference method for mean separation (Muhammad, 1995, p. 252268). Correlation and linear analyses
were used to examine the relationship among different variables and
crop yield by using SYSTAT 12 for Windows (Release 7.0.1, SPSS).

RESULTS AND DISCUSSION


Growth Characteristics
All the data for the growth characteristics of soybean were taken
at the R2 stage, and the results presented here (Table 1) are the average values across 3 yr (20092011). The use of organic and mineral
N sources alone or their combinations, except for WMR100, significantly (p 0.05) increased shoot length, which varied between
62 and 82 cm for Hajira site and 70 to 96 cm for the Rawalakot site
compared with 58 and 62 cm in the controls at each site, respectively
(Table 1). The relative increase in shoot length (over the control)
was 19 to 43% for Hajira and 10 to 49% for the Rawalakot site.
The highest shoot length at both sites was recorded in the UN100
and UN50 + PM50 treatments, and it did not differ between these
two treatments, showing that application of PM with 50% UN
produced plants with a height similar to that recorded for the full
UN100. These results were in accordance with those reported by
Pirdashti et al. (2010), showing that soybean plant height was
the highest for 40 Mg ha1 sewage sludge and vermicomposting enriched with half commercial fertilizer. Sharief et al. (2010)
reported that soybean supplemented with 75% FYM + 25% urea or
75% PM + 25% urea was significantly taller (3 and 5%, respectively)
than the plants amended with 100% urea.
The response of soybean roots to organic or combined treatments
was substantially higher than the response shown by the shoot
(Table 1). The combined applications of UN50 + PM50, UN50 +
WMR50, and UN50 + PM25 + WMR 25 exhibited greater root

length than UN100, and the relative increase over the control was
73 to 109% and over UN100 was 9 to 17%. The results reported
here coincide with the findings of Bandyopadhyay et al. (2010), who
reported that a combined application of NPK and FYM recorded
significantly higher (P < 0.05) root length density and root mass
density of soybean over NPK (28 and 65%, respectively) and the
control (63 and 175%, respectively). This may be attributed to a better nutrient supply and a better physical environment when organic
amendments were combined with mineral N fertilizer. Hati et al.
(2006) also reported that NPK + FYM treated plots maintained
higher root length and root mass density of soybean than the NPK
and control treatments. The effect of different amendments on the
leaf surface area (LSA) showed that the maximum LSA at both sites
was recorded in the treatments receiving UN100 or UN50 + PM50,
while the highest chlorophyll contents were observed in the UN100,
UN50 + PM50, and UN50 + PM25 + WMR 25 treatments.
We speculate that the increase in plant growth due to the combined treatments was largely due to the immediate supply of N at
early growth stages by mineral N sources and to a continued N supply
from organic amendments during the later stages of plant development due to the initial immobilization and then slow mineralization
rates that retained available N in the mineral pool for longer periods
(Adeli et al., 2005; Chaudhary et al., 2014). The correlation between
different growth characteristics, yields, and nutrient uptake is presented in Supplemental Table 4. A significant and positive correlation
existed between growth characteristics and yield components (1000seed weight [TSW], DMY, and seed yield) and nutrient (NPK)
uptake of soybean. Root length also had significant correlations with
the number and dry weight of root nodules and N2 fixation. These
results highlighted the importance of growth characteristics for
nutrient uptake and yields of soybean.

Nodulation
The number of nodules in each plant grown in the control soil
was 30 for Hajira and 76 for the Rawalakot site (Fig. 1), showing
an extensive nodulation with indigenous Bradyrhizobium populations. The variation in nodulation between the two sites may be
due to the variation in indigenous Bradyrhizobium populations in
addition to the differences in soil (physical properties and nutrient
status) and environmental factors (temperature and moisture, i.e.,
rainfall). Among the different amendments, the numbers of nodules
in UN100 were not different than the control (at both sites), while

Table 1. Effect of the integrated use of organic and inorganic N fertilizers on the growth attributes of soybean grown at Hajira and Rawalakot in Azad
Jammu and Kashmir, Pakistan (values are averages of 3 yr: 2009, 2010, and 2011).
Hajira
Rawalakot
Shoot
Root
Leaf surface
Chlorophyll
Shoot
Leaf surface
Chlorophyll
Treatment
length
length
area
content
length
Root length
area
content
g cm2
cm
cm2
g cm2
cm
cm2
Control
57.7 e
25.5 e
36.8 e
11.1 e
64.2 e
26.8 e
42.2 g
14.6 e
PM100
73.9 c
46.2 bc
57.7 c
24.2 ab
81.3 bc
43.8 bc
55.9 de
21.9 c
WMR100
61.6 e
39.1 d
42.8 d
15.6 d
70.3 de
38.4 d
46.9 f
16.3 e
UN100
82.6 a
45.6 bc
62.3 ab
25.1 ab
93.3 a
42.4 cd
62.3 ab
24.4 ab
PM50 + WMR50
68.5 d
42.7 cd
59.4 bc
20.0 c
75.8 cd
39.4 d
53.1 e
19.3 d
UN50 + PM50
81.6 ab
53.4 a
66.0 a
25.5 a
95.6 a
48.2 a
64.1 a
26.5 a
UN50 + WMR50
73.1 cd
51.3 ab
60.4 bc
22.1 bc
87.0 b
46.9 ab
58.0 cd
23.3 bc
UN50 + PM25 + WMR25
77.2 bc
49.9 ab
61.6 b
23.2 abc
71.8 d
46.3 abc
60.6 bc
25.3 ab
5.07
6.35
3.84
3.35
6.26
4.32
3.37
2.47
LSD (p 0.05)
Fertilizers included poultry manure (PM), wheat milling residues (WMR) and urea N (UN). Subscripts indicate the percentage of each fertilizer used in that treatment
(out of 100 kg ha 1).
Means followed by different letters within a column are significantly different based on LSD (0.05) test.

890

Agronomy Journal Volume 107, Issue 3 2015

organic amendments applied alone or combined with UN significantly increased nodule numbers over the control and UN100. Except
UN100, the response of nodule numbers to the different amendments at Hajira was nonsignificant, while at Rawalakot PM100
exhibited the highest number (129) followed by WMR100 (106) and
UN50 + PM50 (102). Nodule mass (dry weight plant1) in response
to the different amendments varied between the two sites (Fig. 1)
and in general was higher at Rawalakot than Hajira. The maximum
nodule mass at Hajira was recorded in the UN50 + PM50 treatment,
which was significantly (P 0.05) higher than that recorded in the
control, UN100, and PM50 + WMR50 treatments but on a par with
the remaining treatments. In contrast, the difference among the
added amendments at the Rawalakot site was significant (except the
control and UN100), and PM100 showed the highest nodule mass followed by UN50 + PM50. These results clearly demonstrate the positive effect of both PM and WMR applied alone or combined with
UN on increasing the number and mass of root nodules.
The increase in nodulation by PM and WMR applied alone or
combined with 50% UN may be due to: (i) the slow release of N
from organic amendments that had no harmful effect on nodulation; (ii) the presence of a sufficient amount of P in the organic
amendments, especially in PM, which may help to increase nodulation in plants; or (iii) better soil physical and chemical properties
and microbial activity in response to organic amendments (Ghosh
et al., 2004). These results were in accordance with the findings of
Ghosh et al. (2004), who reported that the number and mass of
soybean nodules (grown on a deep Vertisol) were decreased with a
higher NPK dose, while application of 75% NPK with FYM, phosphocompost, and PM increased nodule numbers by 34, 30, and 4%
and nodule mass by 37, 23, and 12%, respectively, over the control
treatment. A 30% increase in nodule dry weight was recorded in
soybean plants amended with PM compared with plots without
manure (Chiezey and Odunze, 2009). Similarly, a two-to threefold
increase in nodule number was observed in soybean plants treated
with either carbonized or dried chicken manure (Tagoe et al., 2008).
The correlation between the number and mass of root nodules
vs. the growth, yield and N2 fixation of soybean is presented in
Supplemental Table 4. Nodule number had significant correlations
with root length, nodule dry weight, and N2 fixation, while nodule
dry weight (nodule mass) had significant correlations with root
length, leaf surface area, chlorophyll content, TSW, N2 fixation, and
NPK uptake. Neither the number nor mass of root nodules showed
significant correlations with DMY or seed yield.

Dinitrogen Fixation
The N2 fixation of soybean in response to different amendments
was determined at the R4 stage because previous reports suggested
that the highest rate of N2 fixation occurs at the end of flowering and
during the pod-filling stage (Fabre and Planchon, 2000). The results
indicated that N2 fixation at the two sites varied among treatments
and was highest in PM100 and lowest in the control followed by the
UN100 treatment (Fig. 2). The organic amendments PM, WMR,
and PM + WMR significantly increased N2 fixation compared with
the control, whereas chemical fertilizer (UN100) did not affect N2
fixation. The low N2 fixation in UN100 may be explained by the
inhibitory effect of applied mineral N fertilizer on nodule formation
and activity. In earlier studies, a significant reduction in N2 fixation of
soybean (lower than the control) was observed at each increased level
of fertilizer N, as a result of which Ndfa values declined from 76% at
N0 to 27% at 230 kg N ha1 (Singh et al., 2004). The possibility of
substantial accumulation of NO3N in the soil under the N fertilizer treatment in this research severely suppressed nodulation, thereby
decreasing N2 fixation, consistent with many other studies (Peoples
and Herridge, 1990).
The organic amendments PM100, WMR100, and PM50 +
WMR50 displayed a significant increase in N2 fixation from 30 and
58 kg N ha1 at T0 (control treatments at Hajira and Rawalakot) to
35 to 73 kg N ha1 (Fig. 2). The maximum amount of N2 fixed was
43 kg N ha1 at Hajira, while 73 kg N ha1 was fixed at Rawalakot site
the treatment supplemented with PM100. It has been recognized that
a high level of organic matter buildup in soil due to the addition of
organic amendments improves soil physical conditions and enhances
nutrient levels and microbial activity that eventually increases N2 fixation in soybean (Singh et al., 2004). The integrated use of UN with
PM or WMR in our research improved N2 fixation of soybean, suggesting that continuous use of organic amendments (PM and WMR)
with mineral N had a beneficial effect on N2 fixation.
The maximum N2 fixation values reported here
(4373 kg N ha1) are in accordance with or higher than those
reported in an early maturing soybean line in the southern Guinea
savanna of Nigeria (37 kg N ha1) but lower than a late maturing
soybean line (average of 126 kg N ha1) (Sanginga et al., 1997).
In general, an average of 100 to 111 kg N ha1 N2 fixation has
been reported as appropriate for soybean (Sanginga et al., 1997;
Salvagiotti et al., 2008). However, maximum N2 fixation values of
up to 360 to 450 kg N ha1 have been reported by several researchers
(Rennie et al., 1988; Unkovich and Pate, 2000; Giller, 2001). The
Fig. 1. Effect of poultry manure, wheat straw
residues, urea N, and their combinations on
the number and dry weight of root nodules
of soybean grown at two locations (Hajira
and Rawalakot) in the Poonch district, Azad
Jammu and Kashmir, Pakistan. The vertical
line on each bar represents the LSD (P
0.05) among different treatments: T0, control
without any amendment (unfertilized control);
T1, 100% poultry manure (PM); T 2 , 100%
wheat milling residues (WMR); T3, 100% urea
N (UN); T4, 50% PM and 50% WMR; T5, 50%
UN and 50% PM; T6 , 50% UN and 50% WMR;
T7, 50% UN, 25% PM, and 25% WMR.

Agronomy Journal Volume 107, Issue 3 2015

891

results demonstrate that root nodulation and the indigenous rhizobia


population in the study area were active and had a substantial potential to fix atmospheric N2. However, the correlation between N2 fixation and total N uptake was nonsignificant (r = 0.13), showing that
the contribution of N2 fixation to total N uptake by soybean under
our conditions was not a dominant factor. In contrast to our results,
Salvagiotti et al. (2008) reported a positive relationship between N
uptake and N2 fixation of soybean. It is important to mention that
we determined soybean N uptake at physiological maturity while N2
fixation was determined at the R4 stage, and the nonsignificant relationship observed between N uptake and N2 fixation may be because
of this timing difference.

Yield and Yield Attributes

Fig. 2. Effect of organic and inorganic N amendments applied alone


or integrated in different combinations on the N2 fixation of soybean
grown for 3 yr at two locations, Hajira and Rawalakot, and their
average. The vertical line on each bar represents the LSD (P 0.05)
among different treatments: T0, control without any amendment
(unfertilized control); T1, 100% poultry manure (PM); T 2 , 100% wheat
milling residues (WMR); T3, 100% urea N (UN); T4, 50% PM and 50%
WMR; T5, 50% UN and 50% PM; T6 , 50% UN and 50% WMR; T7, 50%
UN, 25% PM, and 25% WMR.

low N2 fixation recorded in this study highlights the need to adopt


management strategies to improve N2 fixation such as the introduction of new soybean cultivars with high N2fixing potential, the
use of highly effective Bradyrhizobium strains, and adoption of best
management practices. A wide variation in the proportion of N2
fixation by soybean has been observed throughout the world, and
this clearly is due to the diverse climatic and soil conditions, agronomic practices, rhizobial strains, and soybean cultivars used in different parts of the world (Singh and Shivakumar, 2010).
Averaged across the sites, the N2 fixation in the control treatment
was 44 kg ha1, which significantly increased to between 48 and
58 kg ha1 following the application of different amendments (except
UN100), showing a relative increase between 9 and 32%. The maximum average N2 fixation was recorded in PM100 followed by UN50 +
PM25 + WMR25; the difference between the two was significant.
Tagoe et al. (2008) and Chiezey and Odunze (2009) observed maximum nodulation and N2 fixation in soybean with the application of
PM. The highest N2 fixation in the PM or integrated treatments may
be attributed to the supply of organic matter by these amendments
in addition to improvement of soil properties. It has been reported
that the presence of a sufficient quantity of organic matter in soil is
often a major factor contributing to N2 fixation in soybean because it
improves microbial activity (Singh and Shivakumar, 2010).
Total N2 fixation by the plants grown at Hajira was significantly
lower (36 kg ha1) than for those grown at Rawalakot (63 kg ha1).
The relative increase in N2 fixation for Rawalakot was 75% over the
Hajira site. The reasons for higher N2 fixation at Rawalakot may be
better soil physical and chemical conditions (Supplemental Table
2) or may be the higher populations or higher effectiveness of indigenous rhizobia.
To examine the effect of root nodulation on N2 fixation, regression analysis was done between the numbers and mass of nodules
with N2 fixation. By taking the average values of the two sites, regression analysis showed a significant correlation between the number
and mass of nodules and N2 fixation (r = 0.86 and 0.79). These

892

The results presented in Table 2 indicated that the application of N


fertilizers from different sources improved soybean yields compared
with the control. Among the different amendments, WMR100
displayed the lowest TSW, DMY, and seed yields at both sites, while
UN100 showed the highest DMY and seed yield, followed by the
yields under UN50 + PM50. It has been reported that crop residues
with a wider C/N ratio and higher lignin, cellulose, and hemicellulose
were resistant to decomposition and resulted in slow N mineralization
and fast immobilization (Hassan, 2013). The low yields under WMR
in this study are therefore attributed to slow mineralization due to
its high C/N ratio and lignin, cellulose, and hemicellulose contents
(Supplemental Table 3). Zhu (2007) reported that among different
organic materials, rice straw was resistant to microbial decomposition
because of the wide C/N ratio and the high content of lignocellulose.
In contrast to WMR, PM led to a significant increase in soybean
yields compared with the control (25%), WMR100 (17%), and
PM50 + WMR50 (14%). The increasing effect of PM is attributed to
the higher mineralization rate of PM (up to 200 mg N kg1) observed
in our previous study (Abbasi and Khizar, 2012) and the presence of
a higher concentration of essential nutrient elements (Supplemental
Table 3). Tagoe et al. (2008) reported a 30% increase in soybean
yield by the application of PM at a rate equivalent to 100 kg N ha1.
Similarly, Chiezey and Odunze (2009) reported a consistent increase
(33%) in soybean seed yield in 2 yr by the application of PM.
The combined use of organic amendments with one-half of the
total N addition coming from UN resulted in soybean yields significantly higher than the sole application of organic amendments. The
relative increase in seed yield in UN50 + PM50, UN50 + WMR50,
and UN50 + PM25 + WMR 25 was 18, 20, and 27% compared with
the sole application of PM100, WMR100, and PM50 + WMR50,
respectively. The corresponding increase in dry matter yield was 7,
12, and 9%, respectively. Application of UN alone displayed the
highest seed yields at both sites, 1520 and 1460 kg ha1, followed by
the yields recorded under UN50 + PM50, 1385 and 1375 kg ha1.
These results suggested the superiority of UN over organic and
integrated amendments. It also indicated the poor efficiency of the
indigenous Bradyrhizobium population to increase soybean yields
to the level that was achieved by using N fertilizer. The soybean
nodulation, especially under Rawalakot conditions, was substantial
(129 per plant) and N2 fixation was 73 kg N ha1, but the nodulation and N2fixing potential of soybean had not been converted to
higher seed yields. Harper (1974) concluded that neither symbiotic
N2 fixation nor a high soil N supply alone were capable of producing
maximum soybean yields, and maximum soybean yield was possible
only when symbiotic N2 fixation and the uptake of inorganic soil

Agronomy Journal Volume 107, Issue 3 2015

Table 2. Effect of integrated use of organic and inorganic N fertilizer on the yield and yield attributes of soybean grown at Hajira and Rawalakot of
Azad Jammu and Kashmir, Pakistan (values are averages of 3 yr: 2009, 2010, and 2011).
Hajira
Rawalakot
1000-seed
1000-seed
Treatment
weight
Dry matter yield
Seed yield
weight
Dry matter yield
Seed yield
g
kg ha1
g
kg ha1
Control
148 f
1805 g
810 f
161 f
2134 f
1024 f
PM100
171 d
2135 de
1049 d
193 c
2512 d
1246 d
WMR100
162 e
2001 f
878 e
161 f
2240 e
1080 e
UN100
190 b
2465 a
1517 a
203 a
2703 a
1463 a
PM50 + WMR50
167 de
2110 e
897 e
176 e
2286 e
1121 e
UN50 + PM50
199 a
2352 b
1384 b
203 a
2599 bc
1327 b
UN50 + WMR50
178 c
2183 c
1083 d
189 d
2548 cd
1266 d
UN50 + PM25 + WMR25
183 c
2165 cd
1258 c
198 b
2627 b
1300 c
6.24
36.26
37.56
2.10
60.11
37.57
LSD (P 0.05)
Fertilizers included poultry manure (PM), wheat milling residues (WMR) and urea N (UN). Subscripts indicate the percentage of each fertilizer used in that treatment
(out of 100 kg ha 1).
Means followed by different letters within a column are significantly different based on LSD (0.05) test.

N functioned simultaneously. Second, the maximum seed yields


recorded in the present study at Hajira and Rawalakot were 1517
and 1463 kg ha1, respectively, substantially lower than the general
optimum yield found in soybean-growing regions of the world. The
level of yield obtained by adding different amendments showed that
the level of response did not meet this expectation. Therefore, some
management practices need to be adopted to increase the level of
response, e.g., application of improved Bradyrhizobium strains, use
of higher yielding soybean cultivars, optimal timing of sowing, etc.
The results of our study showed that seed yields under our conditions depended on the growth characteristics and N uptake of
soybean. There were significant correlations of shoot length, shoot
dry weight, and root length with seed yield. Similarly, N uptake
also showed a significant correlation with seed yield (r = 0.96)
(Supplemental Table 4). These correlations demonstrated the benefits of N to plant growth and nutrient uptake and its impact on the
seed yield of soybean. In our previous study, these growth characteristics of wheat displayed a significant correlation with wheat grain
yield in soil amended with organic and inorganic N sources (Abbasi
and Tahir, 2012). However, no positive and significant correlations
were observed between seed yield and N2 fixation, nodule number,
and nodule dry weight.

amendments only. The relative increase in N, P, and K uptake by


the integrated treatments (averaged) over the organic amendments
was 23, 20, and 12%, respectively. It has been reported that applying organic and inorganic nutrients together shows synergy and
improved synchronization of nutrient release and uptake by crops
(Mugwe et al., 2009). The response of both N and K uptake to the
combined or integrated treatments compared with UN100 was low.
These results were in accordance with those of Ramesh et al. (2009),
who reported that because the nutrient release from the organic
sources is slow compared with the mineral fertilizers, crops that have
a higher N requirement for optimum production responded better to mineral fertilizers than to organic manures, especially in the
initial stage of development or in the initial years of treatment application. The addition of different amendments (organic, inorganic,
and integrated) significantly (P 0.05) increased total P uptake
by 1.5- to twofold compared with the control. The maximum P
uptake of 20 kg ha1 was recorded in the UN100 and UN50 + PM50
treatments.

Plant Nutrient Uptake


Increased N, P, and K uptake (total shoot + seed) by soybean
treated with organic, inorganic, and combined treatments was
observed (Fig. 3) and could be attributed to increased dry matter
production and N, P, and K concentrations in plant tissue (both in
straw and seeds, data not shown). The relative increase in N, P, and
K uptake due to organic amendments was 16 to 73% for N, 34 to
56% for P, and 30 to 33% for K over the control. This increase probably was due to the buildup of organic matter remaining in the soil
following application for three sequential years and the subsequent
release of NPK throughout the growing season. Yadav (2001) suggested that increases in nutrient uptake in organic-amended plants
could be due to the development of a higher root density, associated
with the improved physical conditions of the soil, which in turn
enhanced the nutrient-absorption capacity of the crop. Among
organic amendments, PM showed superiority over WMR, probably
because of substantial differences in mineralization potential, nutrient concentration, and C/N ratio between the two organic sources.
An integrated use of organic and inorganic amendments significantly increased total NPK uptake compared with organic

Fig. 3. Effect of poultry manure, wheat milling residues, urea N, and


their combinations on total N, P, and K uptake (straw + seed) in
soybean grown at two locations (Hajira and Rawalakot) in the Poonch
district, Azad Jammu and Kashmir, Pakistan, during 2009 to 2011. The
vertical line on each bar represents the LSD (P 0.05) among different
treatments: T0, control without any amendment (unfertilized control);
T1, 100% poultry manure (PM); T 2 , 100% wheat milling residues
(WMR); T3, 100% urea N (UN); T4, 50% PM and 50% WMR; T5, 50%
UN and 50% PM; T6 , 50% UN and 50% WMR; T7, 50% UN, 25% PM,
and 25% WMR.

Agronomy Journal Volume 107, Issue 3 2015

893

Table 3. Effect of integrated use of organic and inorganic N fertilizers on agronomic efficiency of N (NAE), physiological efficiency of N (NPE), and N use
efficiency (NUE) of soybean grown at Hajira and Rawalakot in Azad Jammu and Kashmir, Pakistan (values are averages of 3 yr: 2009, 2010, and 2011).
Treatment
Control
PM100
WMR100
UN100
PM50 + WMR50
UN50 + PM50
UN50 + WMR50
UN50 + PM25 + WMR25
LSD(p 0.05)

Hajira
NAE
NPE
kg kg1

2.4 d
9.8 ab
0.7 e
7.6 bc
7.1 a
11.6 a
0.9 e
6.4 c
5.7 b
10.6 a
2.7 d
7.5 c
4.5 c
9.6 abc
0.35

2.35

NUE
%

25 e
9g
61 a
14 f
54 b
36 d
47 c

NAE
kg kg1

2.2 d
0.6 f
4.4 a
1.0 e
3.0 b
2.4 cd
2.8 bc

3.43

0.38

Rawalakot
NPE

9.0 a
6.7 bc
7.4 ab
7.8 a
5.7 c
7.4 abc
7.6 a

NUE
%

25 d
8f
60 a
12 e
53 b
33 c
36 c

1.82

3.69

Fertilizers included poultry manure (PM), wheat milling residues (WMR) and urea N (UN). Subscripts indicate the percentage of each fertilizer used in that treatment
(out of 100 kg ha 1).
Means followed by different letters within a column are significantly different based on LSD (0.05) test.

Nitrogen Use Efficiency


The NAE of added amendments varied between 0.7 to 7.1 kg kg1
at Hajira and 0.6 to 4.4 kg kg1 at Rawalakot (Table 3). The highest
NAE at both the sites was recorded in UN100, while the lowest NAE
was noted in WMR100. The physiological efficiency at Hajira ranged
between 6.4 to 11.6 kg kg1, while at Rawalakot it varied between 5.7
to 9.0 kg kg1. The highest NPE was recorded in UN100 and PM100
applied alone or combined in different proportions.
The NUE of the applied amendments varied significantly among
the treatments, ranging between 9 and 61% at Hajira and from 8 to
60% at Rawalakot (Table 3). The highest NUE at both the sites was
recorded in the UN100 treatment, while WMR100 showed the lowest. The organic amendments WMR100 and PM50 + WMR50 had
only 9 and 13% NUE, respectively, while PM100 showed a relatively
high efficiency of 25%. Our results were in accordance with those of
Limon-Ortega et al. (2008), who reported that the low NUE under
such conditions may be attributed to the low N contents of organic
substrates and slow rate of N mineralization. The application of
half UN with organic amendments significantly increased NUE
compared with their sole application. The NUE in UN50 + PM50,
UN50 + WMR50, and UN50 + PM25 + WMR25 was 54, 36, and
47% at Hajira and 53, 33, and 36% at Rawalakot, respectively. In
comparison with the UN100 treatment, NUE in the integrated treatments was significantly lower; however, the NUE in UN100 was 60%
while the NUE of UN50 + PM50 was 54% (averaged across years and
sites), and the difference between the two was nonsignificant, showing
that UN50 + PM50 was able to generate NUE comparable to UN100.
These results were in accordance with our previous study conducted
under field conditions on maize (Zea mays L.), where the highest
NUE was observed in the full UN treatment followed by UN +
PM (Abbasi et al., 2010). In contrast, Bandyopadhyay et al. (2010)
reported that the NUE in soybean under NPK + FYM was significantly (P 0.05) higher than NPK and the control.
Most of the studies conducted on the integrated use of organic
and inorganic amendments are based on the application of full NPK
with organic amendments or the organic amendments are applied
on a quantitative basis. The NUE and yield components of a crop
will surely be high when full mineral N or NPK is combined with
organic amendments or when the proportion of mineral N is high
enough compared with the organic N source. We applied N from
different sources, and combinations were made on an N-equivalent
basis at the rate of 100 kg N ha1. Similarly, the proportion of

894

mineral and organic N in the different treatments was arranged at


either 50:50 or 50:25:25. It is suggested that to get a proper comparison of integrated treatments with mineral N fertilizer treatments,
an N-equivalent base or proportion of mineral/organic N should be
considered a major criterion for evaluation.

CONCLUSIONS
Three years of continuous application of PM and WMR greatly stimulated nodulation and N2 fixation of soybean but failed to increase soybean productivity. On the other hand, UN application did not increase
nodulation and N2 fixation but substantially improved the yield and
yield components of soybean. However, the integrated use of 1/2 UN
with PM, i.e., UN50 + PM50, not only stimulated nodulation and N2
fixation but also increased the yield and yield components of soybean.
The average yields recorded in UN50 + PM50 were comparable to the
yields recorded in the UN100 treatment, showing that this combination
can save almost half of the mineral N fertilizer and may have a positive
effect on economic as well as environmental prosperity. Our results
suggested a potential for introducing plant- and animal-derived organic
amendments in mountain agriculture ecosystems to sustain crop yields
and increase the N2 fixation potential of legumes. Because of its topography and environmental conditions, the Himalayan region including
the state of Jammu and Kashmir is most appropriate for legume cultivation. Using indigenous organic substrates as soil amendments will be
useful for sustainable agriculture production systems in this region,
which has a high risk of soil erosion.
ACKNOWLEDGMENTS
This work was supported and funded by the Higher Education
Commission (HEC), Islamabad, Pakistan, via Research Project no. 20-1273
(R&D)/08 under the National Research Program for Universities (NRPU).
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