This article was downloaded by: [University of California, Riverside Libraries]

On: 19 November 2012, At: 10:52

Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,
37-41 Mortimer Street, London W1T 3JH, UK

Plant Biosystems - An International Journal Dealing

with all Aspects of Plant Biology: Official Journal of the
Societa Botanica Italiana
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/tplb20

Synergistic effects of biofertilizer with organic and

chemical N sources in improving soil nutrient status
and increasing growth and yield of wheat grown under
greenhouse conditions
a

M. K. Abbasi & M. Yousra

Faculty of Agriculture, Department of Soil and Environmental Sciences, The University of

Azad Jammu and Kashmir, Poonch, Rawalakot, Pakistan
Accepted author version posted online: 23 May 2012.Version of record first published: 18
Jun 2012.

To cite this article: M. K. Abbasi & M. Yousra (2012): Synergistic effects of biofertilizer with organic and chemical N sources
in improving soil nutrient status and increasing growth and yield of wheat grown under greenhouse conditions, Plant
Biosystems - An International Journal Dealing with all Aspects of Plant Biology: Official Journal of the Societa Botanica
Italiana, 146:sup1, 181-189
To link to this article: http://dx.doi.org/10.1080/11263504.2012.695296

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to
anyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation that the contents
will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should
be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims,
proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in
connection with or arising out of the use of this material.

Plant Biosystems, Vol. 146, Supplement, 2012, pp. 181189

Downloaded by [University of California, Riverside Libraries] at 10:52 19 November 2012

Synergistic effects of biofertilizer with organic and chemical N sources

in improving soil nutrient status and increasing growth and yield of
wheat grown under greenhouse conditions

M. K. ABBASI & M. YOUSRA

Faculty of Agriculture, Department of Soil and Environmental Sciences, The University of Azad Jammu and Kashmir,
Poonch, Rawalakot, Pakistan

Abstract
Biofertilizers have been identified as value-added soil amendments for improving efficiency of applied fertilizers and increasing
soil fertility and crop productivity in sustainable farming system. This study explored the effect of biofertilizer (a mixture of
Pseudomonas, Azospirrillium and Agrobacterium strains) supplemented with organic and mineral N fertilizers on soil properties,
yield, and NPK uptake of wheat (Triticum aestivum L.) grown under greenhouse conditions. The treatments included the
application of fertilizer alone or in different combinations with urea N (UN) and/or poultry manure (PM) added at various
strengths. Results indicated that most of the growth characteristics were higher in treatments receiving biofertilizer
supplemented with UN and full PM (200 mg N kg71). However, treatments under full recommended N (200 mg N kg71)
or UNfull PM had similar effects. Chlorophyll contents were highest in the full UN treatment, followed by the treatment
with UNfullPMbiofertilizer. The latter significantly increased the yield and yield components of wheat compared to full
UN and exhibited superiority over PM. Similarly, NPK uptake in plant shoot was highest in UNfull PMbiofertilizer
compared to full UN or full PM treatments. Furthermore, our results showed that biofertilizer supplemented with PM
improved soil properties, such as organic matter, NPK content and concentration of the micronutrients Fe, Mn, Cu and Zn.
The application of biofertilizer increased the efficiency of both organic and mineral N fertilizer but alone was ineffective in
increasing yield. Thus, biofertilizers could be used as value-added soil amendments by supplementing organic and low
chemical fertilizer rates for improving soil fertility and sustaining crop productivity.

Keywords: biofertilizer, poultry manure, nutrient accumulation, soil amendment, soil nutrient status, Triticum aestivum

Introduction
Among the factors affecting food production, nutrient availability is of prime importance as it affects
key physiological and developmental processes in
plants. Application of chemical or mineral N
fertilizers to soil is the main source of N input and
an indispensable component of agriculture today.
However, in most developing countries the use of
chemical fertilizers has drastically declined as a result
of the energy crisis and increasing costs. Most
chemical N fertilizers are produced by industrial N2
fixation, requiring two units of petroleum for each
unit of N fertilizer produced (Hamdi 1982; Wagner
1997). This is expensive, especially for farmers in less
developed countries. In addition, the continuous use

of chemical fertilizers causes a serious threat to

environmental safety and protection.
In sustainable agriculture systems, it is imperative
to use renewable inputs to maximize ecological
benefits and minimize environmental hazards. The
addition of biofertilizers, i.e. live microorganisms
(bacteria, fungi) known to improve plant growth and
crop productivity, along with organic or mineral N
fertilizers is an effective technique for achieving these
targets (Subba Rao and Dommergues 1998). Rhizobium spp., a group of soil bacteria that fix atmospheric N2 in root nodules of leguminous plants,
were the first biofertilizers identified and have been
used commercially as inoculants for legumes for over
100 years (Kannaiyan 2002). Research in the field of
biofertilizers has resulted in the development of

Correspondence: Prof. Dr. M. Kaleem Abbasi, Department of Soil and Environmental Sciences, The University of Azad Jammu and Kashmir, Poonch,
Rawalakot, Pakistan Tel: 925824960046. Fax: 925824960004. Email: kaleemabbasi@yahoo.com
ISSN 1126-3504 print/ISSN 1724-5575 online 2012 Societa` Botanica Italiana
http://dx.doi.org/10.1080/11263504.2012.695296

Downloaded by [University of California, Riverside Libraries] at 10:52 19 November 2012

182

M. K. Abbasi & M. Yousra

several other kinds of microbial inoculants like,

phosphate solubilizing microorganisms, vesicular
arbuscular mycorrhizae (VAM) and plant growth
promoting rhizobacteria (PGPR) (Delgado et al.
2011). Vessey (2003) defined biofertilizer as a
substance containing living microorganisms which,
when applied to seed, plant surfaces, or soil, colonizes
the rhizosphere or the interior of the plant and
promotes growth by increasing the supply or availability of primary nutrients to the host plant.
Biofertilizers are products containing living cells of
different types of microorganisms, which have an
ability to convert nutrionally important elements, in
particular N and P, from unavailable to available from
through biological process such as N fixation and
solubilization of rock phosphate (Narula et al. 2000;
Cakmakci et al. 2001; Kzlkaya 2008). Many bacterial
species were identified to have N2 fixing properties
including Azotobacter sp., Azosprillum sp., Beijerinckia
sp., etc. (Dobereiner, 1997; Kzlkaya 2008). It is well
known that these bacterial species, in particular those
associated with the plant rhizosphere, are able to exert
a beneficial effect upon plant growth. Therefore, their
use as bio-fertilizers for agriculture improvement has
recently been a focus of numerous studies (Cakmakci
et al. 2001; Narula et al. 2005; El-Sirafy et al. 2006;
Aseri et al. 2008; Kzlkaya 2008; Abbasi et al. 2011).
Many workers have reported that biofertilizers are
more effective when supplemented either with organic
manures or with low rates of chemical N or P
fertilizers. In a pot experiment under greenhouse
condition, the grain and straw yield of wheat was
increased by 97% and 33%, respectively, following the
application of different indigenous and non-indigenous strains of Azotobacter chroococcum and A. chroococcum strain Beijerinck-1901 (Kzlkaya 2008). Zaidi
and Khan (2005) reported that in single inoculation
treatments, A. chroococcum significantly increased the
grain and straw yields of wheat by 197% and 42%,
respectively, relative to the control while dual inoculation with A. chroococcum in association with Pseudomonas striata further enhanced grain and straw yields
by 230% and 50%, respectively. Egamberdiyeva and
Hoflich (2004) reported that application of plant
growth promoting rhizobacteria (PGPR) increased
root and shoot dry weight of cotton to a maximum of
32% and 38%, respectively, while the correspondent
increase in NPK uptake was 13%, 39% and 10%
above the control. In an experiment with wheat
under controlled conditions, application of PGPR
significantly increased plant height, shoot fresh
weight and shoot dry weight (25%, 45%, and
86%, respectively) as well as 1000-grain weight and
grain yield (48 and 59%, respectively) The uptake
of N and P by shoots of these plants was increased
by three-fold, while K uptake was increased by 58%
(Abbasi et al. 2011).

Isolation and characterization of microbial strains

from several crops growing in different environments
has already been carried-out at the Nuclear Institutite of Biotechnology and Genetic Engineering,
(NIBGE) Faisalabad Pakistan (Hameed et al. 2004;
Yasmin et al. 2004; Hafeez et al. 2006). There is
evidence that application of these isolated strains or
biofertilizers increase the efficiency of applied and
native N uptake thereby increasing crop yield and
nutrient accumulation in plant and soil. The present
study was conducted to examine the effect of
application of biofertilizers supplemented with urea
N (UN), poultry manure (PM) or both (UNPM)
on growth, yield and nutrient uptake of wheat and
also on the nutrient status of the soil.

Materials and methods

Experimental procedure and treatments
A pot experiment was conducted in the greenhouse
at the Faculty of Agriculture, Rawalakot Azad
Jammu and Kashmir, Pakistan. Wheat (Triticum
aestivum) variety Inqalab-91 was used as a test
crop to investigate the effects of biofertilizer supplemented with UN, PM and UNPM. The experiment consisted of 12 treatments and 3 replications,
with pots distributed in a completely randomized
design: T1 control; T2 urea N (UN) at 200 mg
N kg71; T3 poultry manure (PM) at 200 mg
N kg71; T4 biofertilizer; T5 UN; T6 UNfull PM; T7 UNPM; T8 UNbiofertilizer;
T9 UNfull PMbiofertilizer; T10 UN
PM biofertilizer; T11 full PMbiofertilizer;
T12 PMbiofertilizer. Nitrogen was applied at
an equivalent rate of 200 kg N ha71. Earthen pots
were used, filled with 12 kg of soil obtained from a
fallow field nearby the Faculty research farm. Soil
used in the study was analyzed before sowing and
identified as sandy loam (55% sand, 31% silt and
14% clay) with an oxidizable organic matter content
of 10.2 g kg71, and a total N content of 0.84 g kg71.
The pH in water was 7.1 and the soil C:N ratio was
12. Available P (0.5M NaHCO3 extractable) and K
(1N NH4OAc extractable) contents were 0.90 and
50.0 mg kg71, respectively. DTPA-extractable Fe,
Cu, Zn and Mn contents were 22.1, 2.0, 2.3 and
14.1 mg kg71, respectively. Urea and poultry manures were incorporated into the soil. A basal dose of P
and K was applied in all pots at the rate of 80 and
70 kg P2O5 and K2O ha71 in the form of single super
phosphate and potassium sulfate, respectively.
Uniform-sized seeds were surface sterilized for
3 min in ethyl alcohol 70%, followed by 3 min in
sodium hypochlorite 3%, and, subsequently, rinsed
six times with sterile water and dried. Surfacesterilized seeds were inoculated by soaking for 1 h

Downloaded by [University of California, Riverside Libraries] at 10:52 19 November 2012

Biofertilizer and wheat growth

in liquid culture medium of biofertilizer (Zaidi and
Khan 2005). Biofertilizer was applied at the rate of
1 kg ha71 as directed by the manufacture. Biofertilizer used in this study was a mixture of Pseudomonas,
Azospirrillium and Agrobacterium strains in a 2:2:1
ratio. Biofertilizer was obtained from the Nuclear
Institute of Biotechnology and Genetic Engineering
(NIBGE), Faisalabad Pakistan. After inoculation,
seeds were air dried and sown immediately. Eight
inoculated seeds were sown in each pot. After
germination, thinning was carried out to leave four
uniform seedlings in each pot. The pots were regularly
irrigated to maintain a proper moisture level of
approximately 60% water holding capacity of the soil.
Agro-morphological and chemical analysis
For growth studies, two plants from each pot were
uprooted, with minimal damage to the root system,
50 days after sowing. The root system was then
washed gently under running tap water to remove the
adhering soil particles and separated from the shoots,
which were oven dried for three days at 708C. Total
root length was measured as described by Farrell
et al. (1993). Shoot length as well as shoot and root
fresh and dry weight were also recorded. The ear
leaf area was determined by measuring width and
length of each ear leaf and multiplying by 0.75
(length6width60.75) (Yi et al. 2006). Chlorophyll
content readings were taken with a handheld dualwavelength meter (SPAD 502, Chlorophyll meter,
Minolta Camera Co., Ltd., Japan). Remaining plants
were harvested at complete maturity at the end of the
study. Spike length and number of spikelets per
spike were recorded before final harvest. In
addition, grain yield per plant and 1000-grain
weight were recorded. Harvested plants were rinsed
with deionized water and oven-dried at 758C for
72 h. The dried shoot tissues were ground and
then digested with concentrated HNO3 for atomic
absorption spectroscopy determination of K. Total
nitrogen content of plants was determined by the
Kjeldahl method (Keeney and Nelson 1982)
whereas total P was determined colorimetrically
by the molybdophosphate method (Murphy and
Riley 1962). At the end of the experiment, soil
samples were collected from each treatment and
available N (NO3N), available-P, available K, the
micronutrients Fe, Mn, Cu and Zn were determined. All parameters were measured according to
Ryan et al. (2001).
Statistical analysis
Data regarding the effect of biofertilizer alone or in
combination with UN, PM and UNPM were
analyzed with analysis of variance using statistical

183

software package Statgraphics (1992). Least significant differences (LSD) were given to indicate
significant differences among different treatments.
A probability level of P  0.05 was considered
significant. The correlation co-efficient between a
pair of means of related traits was determined using
software package SigmaPlot-8.

Results and discussion

Growth characteristics
Different N treatments, applied alone or combined in
different proportions, significantly (P  0.05) increased shoot length, shoot fresh weight and shoot
dry weight of wheat (Table I). Root characteristics, i.e.
length, fresh weight and dry weight, as well as leaf
surface area and chlorophyll content exhibited a
significantly higher response to N application compared to the control. Averaged across different N
treatments, shoot length (39%), shoot dry weight
(34%), root length (23%), root dry weight (139%),
leaf surface area (71%) and chlorophyll content (62%)
were increased relative to the control. Application of
biofertilizer alone (T4) did not result in a significant
growth increase when compared with the control
(Table I). However, plants treated with biofertilizer in
combination either with UNfullPM (T9) or
UNPM (T10) exhibited growth rates comparable to those treated with full UN (T2). Similarly,
application of biofertilizer either with UN (T8) or
with full PM (T11) significantly increased most of the
growth characteristics compared to the treatments
receiving only biofertilizer (T4), UN (T5) or full PM
(T3). With regard to PM effect, application of PM
alone (T3) showed significantly lower values compared to the full UN treatment (T2). However, values
were significantly higher than those obtained with the
UN treatment . Similarly, combined application of
full PM with UN (T6) showed values comparable
to full UN treatment (T2). Our results are in
accordance with previous studies where growth of
cotton, maize and black gram was reported to be
higher in treatments of biofertilizer combined with
organic and chemical fertilizers (Khaliq et al. 2006;
Naveed et al. 2008; Javaid 2009). Zahir et al. (2004)
conducted a series of laboratory experiments with
bacterial strains on wheat and reported an increase in
shoot length (38%), shoot dry weight (36%), root
length (20%) and root dry weight (13%) relative to
the control. Cakmakci et al. (2007) reported a similar
increase in wheat growth due to rhizobial strains.
Effects of root colonization by PGPR on vegetative
growth of Origanum majorana L., showed significant
increases in shoot length, shoot weight, number of
leaves, number of nodes, and root dry weight in
comparison to controls (Banchio et al. 2008).

184

M. K. Abbasi & M. Yousra

(UN). Naveed et al. (2008) reported that grain yield
and 1000-grain weight of maize were, respectively,
13% and 10% higher after treatment with biofertilizer88 kg N ha71 compare with the full dose of N
fertilizer i.e. 175 kg N ha71. In our case, grain yield
and 1000-grain weight were, respectively, 25% and
17% lower with biofertilizerUN than with the full
dose of N fertilizer.
Plant treated with UNbiofertilizer exhibited a
7% increase in 1000-grain weight and a 10% increase
in grain yield compared to plants receiving only
UN. Similarly, application of full PMbiofertilizer
resulted in a 5% and 20% increase in 1000-grain

Downloaded by [University of California, Riverside Libraries] at 10:52 19 November 2012

Yield and yield attributes

Grain yield, spike length, number of spikelets per
spike and 1000-grain weight were significantly
affected by the use of biofertilizer combined with
organic and chemical N fertilizers (Table II). The
highest grain yield (25% higher than full UN) and
1000-grain weight (14% higher than full UN) were
obtained from the treatment receiving biofertilizer
with UN full PM. These results indicated that
the use of biofertilizer and PM combined with half
rate N fertilizer lead to a significant increase in wheat
yield compared to the sole application of full N

Table I. Effect of biofertilizer supplemented with urea N (UN), poultry manure (PM) and UNPM on the growth characteristics of wheat
grown under greenhouse conditions.

Treatments
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
LSD (P  0.05)

Shoot length
(cm)

Shoot fresh
weight
(g plant71)

Shoot dry
weight
(g plant71)

Root length
(cm)

Root fresh
weight
(g plant71)

Root dry
weight
(g plant71)

Leaf surface
area (cm2)

Chlorophyll
content
(mg cm72)

44.5f
70.3a
57.7cd
50.4ef
57.5cd
70.9a
66.0ab
59.6c
67.4ab
66.1ab
63.7bc
52.0de
6.36

7.5f
19.1b
13.4cd
7.6f
9.7ef
21.3b
15.3c
12.3de
25.8a
19.3b
15.7c
11.7de
2.85

2.63c
4.02ab
3.46b
2.47c
2.71c
4.28ab
3.08c
2.87c
4.51a
4.33ab
3.89ab
3.14bc
0.87

12.15f
14.50cde
15.25bc
13.08ef
12.27f
16.12b
14.57cd
15.83bc
17.73a
15.67bc
15.52bc
13.42def
1.43

2.83h
5.90e
6.01de
3.16h
4.55g
6.88bc
6.28cde
5.23f
8.23a
7.25b
6.55cd
5.98de
0.62

0.66f
1.29d
1.39cd
0.84ef
0.97e
1.92b
1.56c
1.60c
2.42a
2.07b
1.89b
1.41cd
0.27

12.6j
25.3bc
19.1efg
13.5ij
16.0hi
24.2cd
20.5ef
17.3gh
33.8a
27.3b
21.5de
18.5fgh
2.76

3.67g
8.29a
5.59cde
3.98fg
5.26de
6.43bc
5.64cde
5.81cd
7.34ab
6.11cd
5.98cd
4.77ef
1.02

*T1 control; T2 urea N (UN) at 200 mg N kg71; T3 poultry manure (PM) at 200 mg N kg71; T4 biofertilizer; T5 UN; T6
UN full PM; T7 UN PM; T8 UN biofertilizer; T9 UN full PM biofertilizer; T10 UN PM biofertilizer;
T11 full PM biofertilizer; T12 PM biofertilizer.

Table II. Effect of biofertilizer supplemented with urea N (UN), poultry manure (PM) and UNPM on the yield and yield components of
wheat grown under greenhouse conditions.
Treatments
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
LSD (P  0.05)

Spike length (cm)

No. of spikelets /spike

1000-grain weight (g)

Grain yield/plant (g)

6.1h
10.5bc
8.5ef
7.0gh
7.9efg
11.1ab
9.8cd
9.1de
11.8a
10.4bc
8.9def
7.7fg
1.21

9d
13bc
14ab
11cd
11cd
16a
14ab
12bc
16a
14ab
14ab
12bc
2.70

34.0f
46.3bc
45.0bcd
37.3ef
39.7de
50.1ab
47.7bc
42.3cde
54.0a
47.7bc
47.3bc
43.3cd
5.62

1.16f
2.41bc
1.67def
1.21ef
1.91cd
2.67ab
2.21bcd
2.11bcd
3.29a
2.49bc
2.08bcd
1.88cde
0.68

*T1 control; T2 urea N (UN) at 200 mg N kg71; T3 poultry manure (PM) at 200 mg N kg71; T4 biofertilizer; T5 UN; T6
UN full PM; T7 UN PM; T8 UN biofertilizer; T9 UN full PM biofertilizer; T10 UN PM biofertilizer;
T11 full PM biofertilizer; T12 PM biofertilizer.

Downloaded by [University of California, Riverside Libraries] at 10:52 19 November 2012

Biofertilizer and wheat growth

weight and grain yield, respectively, compared to
treatment with full PM only. These results demonstrate a substantial increase in the efficiency of both
organic and UN fertilizer when supplemented with
biofertilizer. Shaharoona et al. (2008) reported that
application of rhizobium strains significantly increased the number of tillers per plant (1021%),
grain yield (1543%) and straw yield (2239%) of
wheat. Kumar and Ahlawat (2006) reported that
both Rhizobacteria and Azotobacter caused significant
improvement in growth, yield and N uptake in
wheat. In our experiments, the application of
biofertilizerUN, biofertilizerUNPM and
biofertilizerfull PM resulted in a wheat grain yield
comparable to that obtained with a full UN treatment. Likewise, grain yield after receiving full UN
(T2), i.e. 2.41 g, was equivalent to that obtained with
UNfull PM (2.67 g). These results indicate that
biofertilizerPM or full PM applied in combination
with half the rate of UN fertilizer could substitute
about half of the recommended fertilizer N under
controlled environmental conditions. These results
are in accordance with our previous study on cotton,
where the use of half dose of mineral NPK with
effective microorganisms (EM)organic matter
(OM) saves mineral N fertilizer by almost 50%
compared to a system with only mineral NPK
application (Khaliq et al. 2006).
The increases in yield and yield attributes due to
the application of biofertilizer supplemented with
organic and chemical N fertilizer may be explained
by mechanisms of N2 fixation (Lakshminarayana
et al., 1992), ammonia excretion (Narula et al.
1980), phosphate solubilization (Kundu and Gaur
1980), and growth hormone production (Azcon and
Barea 1976). For example, the Azotobacter culture
used in biofertilizers is reported as a producer of

185

several plant hormones, such as gibberellic acid,

indole 3-acetic acid (IAA), and cytokinin, that
promote growth and yield of a crop (Barea and
Brown 1974; Kzlkaya 2008). In fact, Iruthayaraj
(1981) observed that inoculation with Azotobacter
could increase cotton yield by 1528% due to N2
fixation, and the production of antibacterial and
antifungal compounds, growth regulators and siderophores.
Nutrient accumulation in plant
Application of biofertilizer alone or in combination
with PM and UN significantly increased N, P and K
content in wheat (Table III). For example, application of biofertilizer alone increased NPK content in
wheat shoots from 1.11%, 0.18%, and 1.04% in the
control to 1.17%, 0.20%, and 1.16%, respectively.
However, the response of NPK uptake to biofertilizer
alone was insignificant compared to controls. Generally, both PM and UN when applied alone or in
combination significantly increased NPK uptake in
wheat. NPK uptake after receiving UNfull PM
(T6) showed a 15%, 37% and 11% increase in NPK
uptake compared to full UN treatment (T2). These
results are in accordance with our previous study in
maize (Abbasi et al. 2010) where NPK uptake after
receiving UNPM (UN90PM30) was either
equivalent or higher than full UN (UN120).
Application of UNbiofertilizer (T8) resulted in
a 15%, 14% and 10% increase in NPK uptake,
respectively, compared to UN treatment (T5).
Similarly, biofertilizer applied in combination with
PM (full PMbiofertilizer) (T11) resulted in 19%,
23% and 15% increases in NPK uptake, respectively,
compared to full PM treatment (T3). NPK uptake
also increased in plants treated with PMbiofer-

Table III. Effect of biofertilizer supplemented with urea N (UN), poultry manure (PM) and UNPM on NPK accumulation in wheat grown
under greenhouse conditions.
Treatments
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
LSD (P  0.05)

Shoot N (%)

Shoot P (%)

Shoot K (%)

N-uptake (mg plant71)

1.11g
1.72bc
1.35ef
1.17fg
1.36ef
1.89ab
1.65cd
1.51de
1.97a
1.78abc
1.48de
1.33ef
0.21

0.18d
0.25c
0.32b
0.20cd
0.21cd
0.37ab
0.35b
0.22cd
0.43a
0.36b
0.37ab
0.32b
0.06

1.04g
1.38abc
1.37abcd
1.16f
1.21ef
1.45a
1.32bcde
1.27cdef
1.48a
1.39ab
1.44a
1.26def
0.11

29h
69c
47ef
29h
37g
81b
51de
43fg
89a
77b
58d
42fg
7.26

P-uptake (mg plant71) K-uptake (mg plant71)

5e
10d
11cd
5e
5e
16ab
11cd
6e
19a
16ab
14bc
10d
3.31

28j
55d
47e
29ij
33hi
62b
41f
36gh
67a
60bc
56cd
40fg
4.62

*T1 control; T2 urea N (UN) at 200 mg N kg71; T3 poultry manure (PM) at 200 mg N kg71; T4 biofertilizer; T5 UN; T6
UN full PM; T7 UN PM; T8 UN biofertilizer; T9 UN full PM biofertilizer; T10 UN PM biofertilizer;
T11 full PM biofertilizer; T12 PM biofertilizer.

Downloaded by [University of California, Riverside Libraries] at 10:52 19 November 2012

186

M. K. Abbasi & M. Yousra

tilizer (T12) compared to those treated with UN

(T5). These results demonstrate a substantial increase in the efficiency of both PM and UN fertilizer
when supplemented with biofertilizer. Overall, the
combined application of biofertilizer and UNfull
PM (T9) exhibited the highest NPK, with 22%, 48%
and 17% increases in NPK uptake, respectively,
compare to full UN treatment (T2).
The increase in nutrient accumulation/uptake due
to biofertilizers/PGPR was previously reported for
various crops, i.e. in cotton and pea (Egamberdiyeva
and Hoflich 2004), in wheat (Khan and Zaidi 2007),
and in maize (Wu et al. 2005; Egamberdiyeva 2007).
Both a larger total biomass (shoot dry weight) and a
higher N concentration contributed to the increase in
N accumulation in plants after inoculation with
bacteria. This increase may be explained by mechanisms of N2-fixation, P-solubilization, siderophore
and IAA production of bacterial strains, when
applied with organic and chemical N fertilizer as
reported in other studies (Salantur et al. 2005). Zaidi
and Khan (2005) found a 2794% increase in N
uptake by wheat shoots following the application of
different rhizobial strains and similar responses were
found in P and K uptake. An increase in P-uptake
compared to controls may be due to bacterial
solubilization of insoluble phosphates in the soil.
The rhizosphere of cereal crops was found to be a
harbor of a large number of phosphate solubilizing
bacteria (Narula et al. 2000) that have been showed
to play an effective role in P uptake and growth
promotion (Lifshitz et al. 1987). With regard to the
increase in K uptake due to the application of
biofertilizer, Sheng and He (2006) pointed out that
organic acids, e.g. citric, oxalic, tartaric, succinic

etc., produced by isolated rhizobateria are able to

chelate metals and mobilize K from K-containing
minerals. In addition, the increased nutrient uptake
by plants inoculated with effective strains has been
attributed to the production of plant growth regulators by bacteria present at the root interface, which
stimulate root development, resulting in a better
absorption of water and nutrients from the soil
(Lifshitz et al. 1987). In our experiments, the
increase in nutrient uptake was associated with
higher root length and biomass (Table I). Generally,
the bacterial strains used in biofertilizers are capable
to produce IAA which influence root biomass and
thus increasing the root area available for nutrient
uptake (Sahu and Jana 2000).
Changes in soil properties
Soil analysis (after crop harvest) showed that the
highest OM content was found after treatment with
UNfull PMbiofertilizer (T9), increasing 28%,
compared to the control (Table IV). The total
N content was highest after treatment with full
PMbiofertilizer (T11) showing a 12% increase
compared to the control. Treatments with
UNPM biofertilizer (T10), and full PM
(T11) showed comparable values for these parameters with no significant differences between them.
The Azotobacter bacteria present in biofertilizers are
reported to produce a variety of growth-promoting
substances, including IAA, gibberellins and B
vitamins (Hegde et al. 1999). These substances
stimulate, at least to some degree, the production of
root exudates which were shown to transfer nearly 5
21% of all photosynthetically fixed carbon to the

Table IV. Effect of biofertilizer supplemented with urea N (UN), poultry manure (PM) and UNPM on macro and micronutrients present
in the soil, analyzed after crop harvest.

Treatments

N
(g kg71)

O.M
(g kg71)

C:N
ratio

P
(mg kg71)

K
(mg kg71)

Fe
(mg kg71)

Mn
(mg kg71)

Cu
(mg kg71)

Zn
(mg kg71)

T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
Pre-sowing
LSD (P  0.05)

0.84efg
0.89bcd
0.93ab
0.88cde
0.83fgh
0.90abcd
0.87def
0.79h
0.87def
0.92abc
0.94a
0.80gh
0.84efg
0.0421

9.6de
8.7e
11.7ab
8.8e
7.4f
11.1abc
10.9bc
9.1de
12.3a
11.9ab
11.2abc
11.0bc
10.2cd
1.21

11cd
10de
13ab
10de
9e
12bc
13ab
12bc
14a
13ab
12bc
14a
12bc
1.12

0.90f
1.38f
4.13bc
2.84de
1.58f
5.38a
3.23cd
1.88ef
4.40ab
3.03d
4.91ab
3.13cd
0.90f
1.06

54.0g
60.0f
75.0bc
61.0ef
60.0f
70.0cd
66.0de
63.0ef
82.0a
73.0bc
77.0ab
70.0cd
50.0g
5.41

20.4ef
21.7de
24.5abc
21.2def
19.5f
22.4cde
22.0de
20.5ef
24.9ab
22.8bcd
26.0a
24.7ab
22.1de
2.17

12.2e
13.5cde
15.3ab
13.3cde
13.2de
14.9abc
14.0bcd
14.6abcd
15.8a
13.8bcde
16.0a
13.7bcde
14.1bcd
1.61

1.9f
2.3cd
2.9a
2.2de
2.0ef
2.5bc
2.3cd
2.0ef
2.8a
2.3cd
2.7ab
2.4cd
2.0ef
0.21

1.8f
2.0ef
2.7ab
2.2de
2.2de
2.6bc
2.7ab
2.3d
2.9a
2.4cd
2.9a
2.6bc
2.3d
0.21

*T1 control; T2 urea N (UN) at 200 mg N kg71; T3 poultry manure (PM) at 200 mg N kg71; T4 biofertilizer; T5 UN; T6
UN full PM; T7 UN PM; T8 UN biofertilizer; T9 UN full PM biofertilizer; T10 .UN PM biofertilizer;
T11 full PM biofertilizer; T12 PM biofertilizer.

Downloaded by [University of California, Riverside Libraries] at 10:52 19 November 2012

Biofertilizer and wheat growth

rhizosphere, resulting in a substantial increase of the
soil carbon level (Marschner 1995; Walker et al.
2003). Wu et al. (2005) reported that the OM
content of soil treated with biofertilizer increased by
75% compared to the uninoculated control. Another
important characteristic of Azotobacter is the excretion of ammonia in the rhizosphere in the presence of
root exudates (Narula and Gupta 1986, Wu et al.
2005), which could explain why the combined
treatments of PM and biofertilizer in the present
study resulted in a higher (P  0.05) total N content
in the soil.
The soil used in this experiment is fairly poor in
available P (0.90 mg kg71). However, available P
(Olsen-P) in soil significantly increased when biofertilizer was added to PM (Table IV). Therefore,
use of biofertilizer either with organic manures or
chemical fertilizer can play an important role in
improving P bioavailability. The increase in soil P
content might be due to the P-solubilizing potential
of the isolates used in biofertilizer. Several authors
attribute the production of organic acids, chelating
oxo-acids from sugars, and exchange reactions in the
growth environment to the solubilization of inorganic
insoluble phosphates by microorganisms (Molla and
Chaudhry 1984; Gupta et al. 1994). Soil K content
showed a similar response to different amendments
as that recorded for N and P. Likewise, biofertilizer
in combination with PM significantly increased the
soil micronutrient content. Concentrations of Fe,
Mn, Cu and Zn in soil were highest after application
of full PMbiofertilizer (T11), followed by UNfull PMbiofertilizer (T9), with no significant
difference them. The relative increase in soil micronutrients due to the application of biofertilizer is
attributed to the contribution of microorganisms in
the decomposition of organic wastes and residues
present in the soil or applied through organic
materials, thereby releasing more nutrients from
these substrates in the soil (Javid 2006).
Conclusions
Application of biofertilizer containing beneficial
microbes showed a promoting effect on growth and
nutrients accumulation of wheat as well as significant
improvements in soil properties under greenhouse
conditions. This study indicates some distinct
benefits of the application of biofertilizer combined
with PM and UN fertilizer compared to a full supply
of either PM or UN alone. Our results confirm that
besides increasing crop growth and yield, such
practices save mineral N fertilization which might
positively affect sustainable agricultural production
in soils low in organic matter. In addition, the
possibility of sustaining soil ecology and the environment cannot be ignored. The increased concentra-

187

tion of NPK in plant and soil demonstrate that

application of biofertilizer in combination with PM
and UN increases the efficiency of applied N
fertilizers. Supplementing biofertilizer with organic
N sources or low rates of chemical N fertilizer could
compensate for nutrient deficiency and higher rates
of chemical N fertilization. The use of biofertilizer,
therefore, has the potential to decrease the input
costs of agricultural production, and may be applied
to increase yield potential of low commercial value
sites and to improve the nutrient status and physical
conditions of poor soils.
Acknowledgments
We would like to express our warm gratitude to our
colleagues at the Institute of Natural Resources and
Environmental Sciences, National Agriculture Research Center (NARC), Islamabad, Pakistan for
assisting us in conducting this research, especially
in soil and plant analysis. Thanks to Dr. Sohail
Hameed of the Nuclear Institute of Biotechnology
and Genetic Engineering (NIBGE), Faisalabad
Pakistan, for providing the biofertilizer for this
experiment.

References
Abbasi MK, Khaliq A, Shafiq M, Kazmi M, Ali I. 2010.
Comparative effectiveness of urea N, poultry manure and their
combination on changes in soil properties and maize productivity under rainfed conditions of Rawalakot Azad Jammu and
Kashmir, Pakistan. Exp Agric 46: 211230.
Abbasi MK, Sharif S, Kazmi M, Sultan M, Aslam M. 2011.
Isolation of plant growth promoting rhizobacteria from wheat
rhizosphere and their effect on improving growth, yield and
nutrient uptake of plants. Plant Biosyst 145: 159168.
Aseri GK, Jain N, Panwar J, Rao AV, Meghwal PR. 2008.
Biofertilizers improve plant growth, fruit yield, nutrition, metabolism and rhizosphere enzyme activities of Pomegranate (Punica
granatum L.) in Indian Thar Desert. Sci Hortic 117: 130135.
Azcon R, Barea JM. 1976. Synthesis of auxins, gibberrellins and
cytokinins by Azotobacter vinelandii and Azotobacter beijerinckii
related to effects produced on tomato plants. Plant Soil 43:
609613.
Banchio E, Bogino PC, Zygadlo J, Giordano W. 2008. Plant growth
promoting rhizobacteria improve growth and essential oil yield in
Origanum majorana L. Biochem Syst Ecol 36: 766771.
Barea JM, Brown ME. 1974. Effects of plant growth produced by
Azotobacter paspali related to synthesis of plant growth
regulating substances. J Appl Bacteriol 37: 583593.
Cakmakci R, Erat M, Erdogan U, Donmez MF. 2007. The
influence of plant growth-promoting rhizobateria on growth
and enzyme activities in wheat and spinach plants. J Plant Nutr
Soil Sci 170: 288295.
Cakmakci R, Kantar F, Sahin F. 2001. Effect of N2-fixing bacterial
inoculations on yield of sugar beet and barley. J Plant Nutr Soil
Sci 164: 527531.
Delgado JA, Serrano, JM, Lopez, F, Acosta FJ. 2011. Effects of
fertilization and neighbour removal on biomass allocation and
fruit production in Cistus ladanifer. Plant Biosyst 145: 324330.

Downloaded by [University of California, Riverside Libraries] at 10:52 19 November 2012

188

M. K. Abbasi & M. Yousra

Dobereiner J. 1997. Biological nitrogen fixation in the tropics:

social and economic contributions. Soil Biol Biochem 29: 771
774.
Egamberdiyeva D, Hoflich G. 2004. Effect of plant growthpromoting bacteria on growth and nutrient uptake of cotton
and pea in a semi-arid region of Uzbekistan. J Arid Environ 56:
293301.
Egamberdiyeva D. 2007. The effect of plant growth promoting
bacteria on growth and nutrient uptake of maize in two
different soils. Appl Soil Ecol 36: 184189.
El-Sirafy ZM, Woodward HJ, El-Norjar EM. 2006. Contribution
of biofertilizer and fertilizer N to nutrient uptake and yield of
Egyptian winter wheat. J Plant Nutr 29: 587599.
Farrell RE, Walley FL, Lukey AP, Germida JJ. 1993. Manual and
digital line-intercept methods for measuring root length: a
comparison. Agron J 85: 12331237.
Gupta R, Singal R, Shankar A, Kuhad RC, Saxena RK. 1994. A
modified plate assay for screening phosphate solubilizing
microorganisms. J G Appl Microbiol 4: 255260.
Hafeez FY, Yasmin S, Ariani D, Rahman M, Zafar Y, Malik KA.
2006. Plant growth-promoting bacteria as biofertilizer. Agron
Sustain Dev 26: 143150.
Hamdi YA. 1982. Application of nitrogen-fixing systems in soil
improvement and management. FAO Soils Bull. 49. Rome:
Food and Agriculture Organisation.
Hameed S, Yasmin S, Malik KA, Zafar Y, Hafeez FY. 2004.
Rhizobium, Bradyrhizobium and Agrobacterium strains isolated
from cultivated legumes. Biol Fertility Soils 39: 179185.
Hegde DM, Dwived BS, Sudhakara SN. 1999. Biofertilizers for
cereal production in India a review. Ind J Agric Sci 69: 73
83.
Iruthayaraj MR. 1981. Let Azotobacter supply nitrogen to cotton.
Intensive Agric 19: 2327.
Javaid A. 2009. Growth, nodulation and yield of black gram [Vigna
mungo (L.) Hepper] as influenced by biofertilizers and soil
amendments. Afr J Biotechnol 8: 57115717.
Javid A. 2006. Foliar application of effective microorganisms on
pea as an alternative fertilizer. Agron Sust Dev 26: 257262.
Kannaiyan S. 2002. Biofertilizers for sustainable crop production.
In: Kannaiyan S, editor. Biotechnology and biofertilizers.
Dordrecht, the Netherlands: Kluwer Academic Publishers.
pp. 949.
Keeney DR, Nelson DW. 1982. Nitrogen-inorganic forms. In:
Page AL, Miller RH, Keeney DR, editors. Methods of soil
analysis. Part 2. Chemical and microbiological properties.
Madison, WI: Soil Science Society of America. pp. 643693.
Khaliq A, Abbasi MK, Hussain T. 2006. Effect of integrated use of
organic and inorganic nutrient sources with effective microorganisms (EM) on seed cotton yield in Pakistan. Bioresour
Technol 97: 967972.
Khan AM, Zaidi A. 2007. Synergistic effects of the inoculation
with plant growth-promoting rhizobacteria and an arbuscular
mycorrhizal fungus on the performance of wheat. Tur J Agric
For 31: 355362.
Kzlkaya R. 2008. Yield response and nitrogen concentrations of
spring wheat (Triticum aestivum) inoculated with Azotobacter
chroococcum strains. Ecol Eng 33: 150156.
Kumar V, Ahlawat IPS 2006. Effect of biofertilizer and nitrogen
on wheat (Triticum aestivum) and their after effects on
succeeding maize (Zea mays) in wheat-maize cropping system.
Ind J Agric Sci 76: 465468.
Kundu BS, Gaur AC. 1980. Estabilshment of nitrogen fixing and
phosphate solubilizing bacteria in rhizosphere and their effect
on yield and nutrient uptake of wheat crop. Plant Soil 57: 223
230.
Lakshminarayana K, Narula N, Hooda IS, Faroda AS. 1992.
Nitrogen economy in wheat (Triticum aestivum L.) through use
of Azotobacter chroococcum. Ind J Agric Sci 62: 7576.

Lifshitz R, Kloepper JM, Kozlowski M, Simonson C, Carlso J,

Tipping EM, et al.
1987. Growth promotion of
canola (rapeseed) seedlings by a strain of Pseudomonas
putida under gnotobiotic conditions. Can J Microbiol 33:
390-397.
Marschner H. 1995. Mineral nutrition of higher plants. 2nd edn.
London: Academic Press.
Molla MAZ, Chaudhry AA. 1984. Microbial mineralization of
organic phosphate in soil. Plant Soil 78: 393399.
Murphy J, Riley JP. 1962. A modified single method for the
determination of phosphate in natural waters. Anal Chem Acta
27: 3136.
Narula N, Gupta KG. 1986. Ammonia excretion by Azotobacter
chroococcum in liquid culture and soil in the presence of
manganese and clay minerals. Plant Soil 93: 205209.
Narula N, Kumar V, Behl RK, Deubel A, Gransee A, Merbach
W. 2000. Effect of P-solubilizing Azotobactor chroococcum
on N,P,K uptake in P-responsive wheat genotypes grown
under greenhouse conditions. J Plant Nutr Soil Sci 163:
393398.
Narula N, Kumar V, Singh B, Bhatia R, Laxminarayana K. 2005.
Impact of biofertilizers on grain yield in spring wheat under
varying fertility conditions and wheat-cotton rotation. Arch
Agron Soil Sci 51: 7989.
Narula N, Lakshminarayana K, Tauro P. 1980. Ammonia excretion
by Azotobacter chroococcum.Biotechnol Sci 23: 467470.
Naveed M, Khalid M, Jones DL, Ahmad R, Zahir ZA. 2008.
Relative efficacy of Pseudomonas spp., containing ACCdeaminase for improving growth and yield of maize (Zea mays
L.) in the presence of organic fertilizer. Pak J Bot 40: 1243
1251.
Ryan J, Estefan G, Rashid A. 2001. Soil and Plant Analysis
Laboratory Manual. International Center for Agricultural
Research in the Dry Areas (ICARDA), Syria.
Sahu SN, Jana BB. 2000. Enhancement of the fertilizer value of
rock phosphate engineered through phosphate-solubilizing
bacteria. Ecol Eng 15: 2739.
Salantur A, Ozturk A, Akten S, Sahin F, Donmez F. 2005. Effect
of inoculation with non-indigenous and indigenous rhizobacteria of Erzurum (Turkey) origin on growth and yield of spring
barley. Plant Soil 275: 147156.
Shaharoona B, Naveed M, Arshad M, Zahir ZA. 2008. Fertilizerdependent efficiency of Pseudomonads for improving growth,
yield, and nutrient use efficiency of wheat (Triticum aestivum
L.). Appl Microbiol Biotechnol 79: 147155.
Sheng XF, He LY. 2006. Solubilization of potassium-bearing
minerals by a wide-type strains of Bacillus edaphicus and its
mutants and increased potassium uptake by wheat. Can J
Microbiol 52: 6672.
StatGraphics Manugistics Inc. 1992. Statistical Graphics System
by Statistical Graphics Corporation, Reference Manual, Version 6. Rockville, MD: Manugistics, Inc.
Subba Rao NS, Dommergues YR. 1998. Microbial Interactions in
Agriculture and Forestry. USA: Science Publishers, Inc. pp. 278.
Vessey JK. 2003. Plant growth promoting rhizobacteria as
biofertilizer. Plant Soil 255: 571586.
Wagner GM. 1997. Azolla: A review of its biology and utilisation.
Bot Rev 63: 126.
Walker TS, Bais HP, Grotewold E, Vivanco M. 2003. Root
exudation and rhizosphere biology. Plant Physiol 132: 4451.
Wu SC, Caob ZH, Lib ZG, Cheunga KC, Wonga MH. 2005.
Effects of biofertilizer containing N-fixer, P and K solubilizers
and AM fungi on maize growth: a greenhouse trial. Geoderma
125: 155166.
Yasmin S, Bakar MAR, Malik KA, Hafeez FY. 2004. Isolation,
characterization and beneficial effects of rice associated plant
growth promoting bacteria from Zanzibar soils. J Basic
Microbiol 44: 241252.

Biofertilizer and wheat growth

Downloaded by [University of California, Riverside Libraries] at 10:52 19 November 2012

Yi ZX, Wang P, Zhang HF, Shen LX, Liu M, Dai MH. 2006.
Effects of type and application rate of nitrogen fertilizer on
source-sink relationship in summer maize in north China plain.
Plant Nutr Fert Sci 12: 294300.
Zahir ZA, Arshad A, Frankenberger WT. 2004. Plant growth
promoting rhizobacteria: Applications and perspectives in
agriculture. Adv Agron 81: 97168.

189

Zaidi A, Khan MS. 2005. Interactive effect of rhizotrophic

microorganisms on growth, yield, and nutrient uptake of
wheat. J Plant Nutr 28: 20792092.