World Applied Sciences Journal 16 (2): 213-219, 2012

ISSN 1818-4952
IDOSI Publications, 2012

Response of Wheat Growth Parameters to Co-Inoculation

of Plant Growth Promoting Rhizobacteria (PGPR)
and Different Levels of Inorganic Nitrogen and Phosphorus
Zahra Saber, Hemmatollah Pirdashti, Mohammadali Esmaeili,
Arasto Abbasian and Ayob Heidarzadeh
Department of Agronomy and Plant Breeding,
Sari Agricultural Sciences and Natural Resources University, Iran
Abstract: N-fixing and P-solubilizing bacteria playing a significant role as a plant growth-promoting
Rhizobacteria (PGPR) in diverse crops. In order to evaluate efficiency of PGPR plus nitrogen and phosphorous
chemical fertilizers on yield of wheat (cv. N-80-19) a field experiment was arranged in split-split plots based on
randomized complete block design with three replications. The main and sub plot consisting of phosphorous
(0, 25 and 50 kg ha 1) and nitrogen (0, 50 and 100 kg ha 1), respectively and sub sub-plot were considered PGPR
at four levels (control, inoculation with nitrogen fixing bacteria (PFB), phosphate solubilizing bacteria (PSB) and
dual inoculation with PFB and PSB). Results showed that application of biofertilizers could increase grain yield
and harvest index as much as 46.6 and 48.8% compared to control, respectively. Integrated treatments showed
better performance in terms of grain number per spike, tiller number and shoot length by 35.57, 35.1 and 31.9%
compared to separate treatments, respectively. Furthermore, biofertilizers significantly reduced P and N fertilizer
application without any reduction in wheat yield related parameters. Generally, results of this study revealed
that using of biofertilizers especially dual inoculation had effective and significant role in terms of all studied
yield characteristics except spike length in given wheat cultivar.
Key words: Biofertilizer

Wheat

Nitrogen

Phosphorous

INTRODUCTION

PGPR

rhizosphere of many plants and are known to colonize the

rhizosphere of wheat [3]. Furthermore, bacterial inoculants
are able to increase plant growth and germination rate,
improve seedling emergence and protect plants from
disease [4]. Phosphate solubilizing bacteria (PSB) are used
as biofertilizer since 1950s [5]. These microorganisms
secrete different types of organic acids e.g., carboxylic
acid [6], reduce the pH in the rhizosphere [7] and
consequently dissociate the bound forms of phosphate
like Ca3 (PO4)2 in calcareous soils. Use of these
microorganisms as environment friendly biofertilizer helps
to reduce the much expensive phosphatic fertilizers.
Phosphorus biofertilizers not only could increase the
availability of accumulated phosphate by solubilization
but also improve efficiency of biological nitrogen fixation
and increase the availability of Fe, Zn and so on, through
production of plant growth promoting substances [8].
The growth promoting effects of Rhizobacteria may
include phytohormone production, N2 fixation and more

Nitrogen and phosphorus are known to be essential

nutrients for plant growth and development, also they are
essential nutrients required by both plants and
microorganisms. The major physiological roles of these
elements are the accumulation and release of energy
during cellular metabolism, Biofertilizers, however, are
well recognized as an important component of integrated
plant nutrient management for sustainable agriculture
and hold a great promise to improve crop yield [1]. A
group of biofertilizers termed plant growth promoting
Rhizobacteria (PGPR) consisting of strains from genera
such as Pseudomonas, Azospirillium, Azotobacter,
Bacillus, Burkholderia, Enterobacter, Rhizobium,
Erwinia and Flavobacterium [2]. Indeed, they produce
metabolites such as plant growth regulators that directly
promote growth and facilitate nutrient uptake by plants.
These bacteria are an important component of the

Corresponding Author: Zahra Saber, Department of Agronomy and Plant Breeding, Sari Agricultural Sciences and Natural
Resources University, Iran.

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World Appl. Sci. J., 16 (2): 213-219, 2012

Table 1: Some characteristics of the soil used
Soil type

Clay
Silt
Sand
--------------------%---------------------

Silt-clay

50

43.23

EC dS/m

pH

C
N
------------%----------

K
P
------(mg/kg soil)-----

2.33

7.15

2.92

320

6.7

0.17

12.32

efficient use of nutrients [9]. Significant increases by

PGPR in growth and yield of agronomical important
crops in response to inoculation with PGPR have been
reported [10-12], Also Azospirillum, Pseudomonas and
Azotobacter strains could affect seed germination and
seedling growth [13]. Recently many researches have
been done in north of Iran conditions and it seems that
these organisms are important for agriculture in order to
promote recycling of plant nutrients and reduce the need
from chemical fertilizers. The objective of present
investigation was to study the effect of single and dual
inoculations with N2-fixing and P-solubilizing bacterial
species as biofertilizer on yield and yield components of
wheat.

Some physical and chemical parameters of soil are

shown in Table 1. Half of nitrogen fertilizers and all
phosphorus fertilizer were applied at sowing time and
half of N was applied at tillering stage. At the end of
experiment plants were randomly hand-harvested from
1 m2 area of unsampled center of each plot to estimate the
plant height, biomass, shoot fresh and dry weights, tillers
number, grain yield, 1000 grain weight, grain number per
spike, shoot length and grain yield. Seeds were carefully
taken from each plant, cleaned and weighed. Yield was
calculated on a per square meter basis and then harvest
index (HI) was determined by the following equation [14]:

MATERIALS AND METHODS

Statistic Analysis: The data were subjected to analysis of

variance (ANOVA), using SPSS 13.0 statistical program.
Mean values were separated according to LSD test at
P=0.05.

HI =

Seed Priming: Seeds of wheat (cv. N-80-19) were

prepared from Sari Agricultural Research Center. N-80-19
is a modern cultivar and grown for its high yield and
quality. After removing the trashes the seeds sterilized for
1 min in 70 % ethanol and then treated in 5 % Na
hypochloride solution for 40 min, followed by rinsing six
times with autoclaved distilled water. Seeds were dried in
room temperature and primed by phosphate solvent
bacteria (PSB), Barvar phosphate 2 by Green
Biotechnology Company, consist of Bacillus lentus,
Pseudomonas putida and nitrogen fixing bacteria (PFB),
Supernitroplast,
consist
of
Bacillus
subtilis,
Pseudomonas fluorescens, Azospirillum spp. which
known as plant growth promoting rhizhobacteria.
Uninoculated seeds considered as control treatments.
Finally, primed seeds of wheat were prepared for sowing.

Seed yield
100
Biological yield

RESULTS
Results of variance analysis are presented in Table 2.
The most of studied traits such as tiller number, harvest
index, shoot length, grain yield, 1000 grain weight, grain
number per spike, biological yield except spike length,
were significantly different at 1% and 5% probability
levels (Table 2). According to the results, all parameters
except spike length increased significantly by inoculation
treatment and the most increase was recorded when dual
inoculation (PFB+PSB) treatment was applied.
Tiller Number: Based on the results of variance analysis
tiller number was significantly affected at 1% probability
levels by phosphorus, nitrogen, biofertilizer and
interaction between phosphorus and nitrogen chemical
fertilizers while interaction between other treatments were
not significant (Table 2). Accordingly maximum tiller
number was recorded from dual inoculation (PFB+ PSB)
seeds with 100 kg ha 1 nitrogen, 25 and 50 kg ha 1
phosphorus (Table 3).

Field Experiment: Field experiment was performed at Sari

Agricultural Sciences and Natural Resources University
of Iran during 2009. The experiment was conducted in
split-split plot based randomized complete block design
with three replications. Levels of phosphorus (0, 25 and
50 kg ha 1) and nitrogen (0, 50 and 100 kg ha 1)
considered as main and subplots, respectively and
sub-sub-plots were PGPR at four levels (uninoculated
control, inoculation with nitrogen fixing bacteria (PFB),
phosphate
solubilizing bacteria (PSB) and dual
inoculation with PFB and PSB). Seeds were sown in 5 m
2 m plots on 22 November in 2009 at experiment farm site.

Grain Yield: Grain yield was markedly influenced by

phosphorus, nitrogen; biofertilizer and all interaction
between treatments. In grain yield attribute, application of
100 kg ha 1 nitrogen and 25, 50 kg ha 1 phosphorus led to
214

World Appl. Sci. J., 16 (2): 213-219, 2012

Table 2: Mean squares from analyses of variance of data for yield and yield component of wheat under farm condition
S.O.V.

df

Tiller

Grain

1000

Grain number

Spikelet number

Shoot

Spike

Total

Harvest

number

yield

seed weight

per spike

per spike

length

length

biomass

index

Replication (R)

1.39ns

149.08ns

0.77ns

0.13ns

0.53ns

0.61ns

0.01ns

800.59ns

1.19ns

Phosphorus (A)

55.29**

49095**

160.7**

789.4**

135.2**

1014**

0.0002ns

5518.3**

1712**

Nitrogen (B)

134.8 **

38014**

127.6**

161.6**

59.5**

3327**

0.03ns

17915**

1404**

Biofertilizer (C)

17.78**

34579**

176.6**

117.4**

15.92**

451.2**

0.64ns

997.5*

98.90**

AB

5.27**

47440**

9.48*

22.32**

6.04**

9.55**

0.003ns

219.5ns

164.9**

AC

0.11ns

4048.2**

1.39ns

0.63**

0.29ns

71.31**

0.06ns

2011**

8.08**

BC

0.21ns

750.37**

4.21**

1.06**

0.21ns

17.4**

0.04ns

127.7ns

2.36**

ABC

12

0.37ns

248.83**

0.46ns

0.42**

1.23*

5.51**

0.03ns

505.3ns

1.002*

Error

54

0.22

57.43

0.623

0.13

0.56

14.45

0.40

343.17

0.889

11.37

5.25

3.90

6.81

4.17

6.32

5.49

15.67

3.38

CV

*, ** = Significant at 0.05, 0.01 levels, respectively ns = Non-significant

Table 3: Interaction between phosphorus and nitrogen
Phosphorus

Nitrogen

Tiller

Shoot

Grain

1000 grain

Harvest

Spikelet

(kg ha 1)

(kg ha 1)

number

length (cm)

yield (gr.m 2)

Weight (gr)

index

number per spike

1.66

74.75

520.33

44.88

29.16

50

2.50d

25

50

89.40b

549.08f

44.82a

30.78e

15.09

Grain number
per spike

35.68f

15.83d

40.89e
42.40d

100

4.00b

89.83b

584.16e

42.24b

33.31d

16.18d

3.00c

82.08c

595.16d

44.63a

33.67cd

16.93c

46.47c

50

4.08b

99.40a

793.75b

44.68a

44.90b

19.66b

47.29b

100

7.41a

99.88a

866.58a

42.47b

50.24a

20.12ab

49.50a

3.00

82.25

602.66

44.82

34.10

17.33

46.51c

50

4.20

99.65

793.66

42.13

44.89

19.83

ab

47.35b

100

7.50

99.87

868.83

42.68

50.37

20.41

49.47a

c
b
a

Values followed by different letters in a column were significantly different (P < 0.05) using LSD test

Spikelet Number Per Spike: Based on the results of

variance analysis spikelet number per spike was affected
by all simple effects and interaction between nitrogen and
phosphorus fertilizers (Table 2). Spikelet number per spike
was improved by biofertilizer inoculation. Maximum
spikelet number was recorded in plants treated with dual
inoculations (PFB+ PSB) both in 100 kg ha 1 nitrogen and
25 or 50 kg ha 1 phosphorus treatment while minimum
spikelet was observed in control (Table 3, 4, 5).

maximum grain yield (Table 3). However, biofertilizers

(PFB+ PSB) showed significant effect especially on yield
of wheat (Table 5).
1000 Grain Weight: Based on the results of variance
analysis 1000 grain weight was significantly different in
response to different fertilizer treatments (Table 2).
Accordingly 1000 grain weight was improved as a result
of biofertilizer treatment. Maximum 1000 grain weight was
recorded from dual inoculated (PFB+ PSB) in either 0 and
50 kg ha 1 nitrogen or 0 and 25 kg ha 1 phosphorus
treatment while minimum 1000 grain weight was observed
in control treatment (Table 3, 4, 5).

Shoot Length and Spike Length: Results of ANOVA

showed a statistically significant difference shoot length
between treatments overall. By contrast, spike length was
not significantly affected by any treatments (Table 2).
The experiment revealed that shoot length highly
stimulated by seed inoculation with all given bacteria,
especially in mixed inoculation (Tables 3, 4, 5), while in
terms of spike length no significant effect was observed
in the present study (Table 2).

Grain Number Per Spike: Grain number per spike was

affected significantly by simple effect of phosphorus,
nitrogen, biofertilizer and interaction between all
treatments (Table 2). The data indicated that (PFB+ PSB)
treatment significantly increased grain number per
spike compared to control so most effective treatment
was maximum level of nitrogen along with dual
inoculation of PFB+ PSB compared to uninoculant
treatment (Table 5).

Biomass and Harvest Index: According to the results of

variance analysis, biomass yield and harvest index
affected by all simple and interaction effects of given
215

World Appl. Sci. J., 16 (2): 213-219, 2012

Table 4: Interaction between phosphorus and biofertilizer
Phosphorus (kg ha 1)

Biofertilizer

Control
PSB
PFB
PSB+PFB
Control
PSB
PFB
PSB+PFB
Control
PSB
PFB
PSB+PFB

25

50

Grain yield (gr.m 2)

534.33f
566.33e
554.11e
560.00e
678.66d
778.33ab
769.66c
780.66ab
684.77d
776.77abc
775.22bc
783.44a

Total biomass (gr.m 2)

1790.5a
1773.8ab
1768.1bc
1764.6bc
1734.4d
1748.2cd
1761.8bc
1766.8bc
1734.4d
1748.2cd
1761.8bc
1766.8bc

Shoot length (cm)

Harvest index

Grain number per spike

74.33e
86.77d
86.33d
91.21c
90.77c
93.82b
93.66b
96.90a
90.77c
93.78b
93.70b
97.44a

29.84e
31.38d
31.37d
31.75d
39.20c
44.57a
43.73b
44.26ab
39.54c
44.48a
44.04ab
44.42a

36.88f
40.28e
40.23e
41.22d
44.82c
48.23b
47.96b
50.01a
44.70c
48.31b
48.12b
50.00a

Values followed by different letters in a column were significantly different (P < 0.05), using LSD test. PSB, phosphate solubilizing bacteria. PFB, nitrogen
fixing bacteria
Table 5: Interaction between nitrogen and biofertilizer
Nitrogen (kgha 1)

Biofertilizer

Control
PSB
PFB
PSB+PFB
Control
PSB
PFB
PSB+PFB
Control
PSB
PFB
PSB+PFB

50

100

1000 seed weight (gr)

Grain number per spike

40.50
44.78a
44.75a
44.66a
40.39d
44.31a
44.56a
44.94a
39.32e
41.67c
41.82bc
42.52b
d

39.50
43.66g
43.41g
44.98e
42.37h
45.72d
45.71d
46.91c
44.53f
47.45b
47.18bc
49.32a
i

Shoot length (cm)

72.66
80.11e
79.66e
86.33d
91.55c
96.95b
96.74b
99.37a
91.66c
97.44b
97.16b
99.84a
f

Grain yield (gr.m 2)

533.88g
587.00e
578.66f
591.33e
646.88d
733.33b
731.55b
736.88b
717.00c
791.11a
788.77a
795.88a

Harvest index
30.29f
33.21d
32.52e
33.22d
36.73c
41.52b
41.13b
41.38b
41.56b
45.69a
45.49a
45.83a

Values followed by different letters in a column were significantly different (P < 0.05) using LSD test. PSB, Phosphate Solubilizing Bacteria. PFB, nitrogen
fixing bacteria

comparable to those of Boddy et al. [15] who

demonstrated application of bacterial inoculants as
biofertilizers has improved growth and yield of cereal
crops. Minimum tiller number was recorded in
uninoculated control, compared to all other combinations;
these findings are supported by Marcos et al. [16] and
Naveed et al. [17].
Grain yield is one of the significant parameters
between yield and yield components and usually
characterized by biotic and abiotic environment factors
after genetic properties, therefore, the mentioned results
for grain yield was in agreement with the findings of
Dobbelaere et al. [18, 19] who assessed the inoculation
effect of PGPR Azospirillum brasilense on growth of
spring wheat. They observed that inoculated plants had
more grain yield. There are a lot of results indicated that
inoculated plants had more grain yield [20-22]. Also
present results showed that integrated application of
fertilizers (biological and chemical fertilizers) markedly
increased yield and its components. Amujoyegbe et al.

treatments (Table 2). The maximum harvest index was

recorded in 100 kg ha 1 nitrogen and 25 or 50 kg ha 1
phosphorus treatment. This response was probably due
to higher grain yield and biomass. Also interactions
between phosphorus and biofertilizer showed that 25 and
50 kg ha 1 phosphorus and control treatment indicated
the lowest performance for total biomass (Table 4).
Meanwhile, total biomass of wheat was better in
inoculation treatments than control and mineral fertilizer
(50 and 25 kg P/ha) treatments. Hence, the lowest harvest
index values were recorded in the control with 0 nitrogen
treatment, while the highest value was obtained from the
mixed PGPR inoculations, PSB and PFB with mineral
nitrogen (100 kg ha 1) fertilizer treatment (Table 5).
DISCUSSION
All inoculations and fertilizer applications markedly
improved grain yield, total biomass and tiller number of
wheat compared to uninoculated control. The results are
216

World Appl. Sci. J., 16 (2): 213-219, 2012

[23] and Rizwan et al. [24] reported that application of

chemical fertilizer plus biological fertilizer could produce
highest yield compared to either chemical or biological
treatments alone.
1000 grain weight can vary from one crop to another,
between varieties of the same crop and even from year to
year or from field to field of the same variety [25], our
findings were supported by Yasari and Patwardhan [21]
who reported that application of Azotobacter and
Azospirillum strains increased canola yield and weight of
1000 grain. Similarly, our findings in case of 1000- grain
weight were also in accordance with the findings of
Hussain et al. [26] and Zahir et al. [27]. Ibeawuchi and
Onweremalu [28] in an experiment showed that, at first
of all the highest 1000 grain weight was in integrated
fertilizer treatments and the following was in chemical
fertilizer.
Grain numbers per spike are interrelated yield
components [25]. Dobbelaere et al. [19] assessed the
inoculation effect of PGPR Azospirillum brasilense on
growth of spring wheat. They observed that inoculated
plants had more grain number per spike and grain yield.
Similarly, promotion in growth parameters and yields of
various crop plants in response to inoculation with PGPR
were reported by other workers (for example: 4 and 29).
The number of spikelets per spike is one of the
physical components of grain yield which is the potential
number of seeds per spike [25]. Our results confirmed the
previous findings [30, 31, 32, 16, 17], who reported
increased spikelet per spike of various crop plants by
microbial inoculation.
Also, similar results for plant shoot length were
observed in different crops inoculated with Pseudomonas,
Azospirillum and Azotobacter strains [33-35]. Meanwhile,
Burd et al. [36] reported that plant growth promoting
rhizobacteria might improve plant height and productivity
by synthesizing phytohormones, enhance the general
availability of nutrients. These results confirmed the
previous findings [30, 31, 32, 16, 17], who reported
increased plant height of various crop plants by microbial
inoculation. The importance of this effect was reported in
previous researches for component traits like plant height
and spike length [37], while in terms of spike length no
significant effect was observed in the present study.
In term of biomass our results are similar with the
finding of Dobbelaere et al. [18] who observed that
inoculated wheat plants improved germination rate, seed
yield and biomass of root system and shoot biomass.
In addition, there were related results have been reported

by Hflich et al. [38] in barley, Chabot et al. [9] in maize.

Similarly, trials with PSB indicated total yield increases in
rice [20] and maize [39]. Also dual inoculation clearly
increased yields in sorghum [22], barley [40] and wheat
[41].
CONCLUSION
In the current study, the given results supported
possibility of reducing chemical fertilizer using up to 50%
with applying PGPR. Moreover, there were no additional
improvements in most mentioned traits by using high
levels of phosphorus rather than PGPR plus lower levels
of fertilizer. Inoculation of PGPR increased the most
parameters determined in field experiment. The stimulatory
effects of PGPR on the yield and growth of wheat are in
support in diverse crops such as barley and wheat
[42, 43]. The present study indicated that new technology
of dual inoculation of PFB and PSB has advantage over
traditional mineral fertilizers. It is concluded that mixed
inoculants (PSB+PFB) performed better than single isolate
inoculants. However PFB and/or PSB inoculation without
chemical fertilizer was not effective, it seems that adding
biofertilizers and low input of chemical fertilizers would
give promising results. Thus this approach could be
reduce overapplication of P fertilizer for the profit of
farmers and ensure environment-friendly practices.
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