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Isolation of plant growth promoting rhizobacteria from wheat rhizosphere

and their effect on improving growth, yield and nutrient uptake of plants

M. K. Abbasia; S. Sharifa; M. Kazmia; T. Sultanb; M. Aslamb

a
Department of Soil and Environmental Sciences, Faculty of Agriculture, University of Azad Jammu
and Kashmir, Rawalakot, Azad Jammu and Kashmir, Pakistan b Soil Biology and Biochemistry Section,
National Agriculture Research Center, Islamabad, Pakistan
Online publication date: 03 March 2011
To cite this Article Abbasi, M. K. , Sharif, S. , Kazmi, M. , Sultan, T. and 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 Biosystems - An International Journal Dealing with all Aspects of Plant Biology, 145: 1, 159 168
To link to this Article: DOI: 10.1080/11263504.2010.542318
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Plant Biosystems, Vol. 145, No. 1, March 2011, pp. 159168

Isolation of plant growth promoting rhizobacteria from wheat

rhizosphere and their effect on improving growth, yield and nutrient
uptake of plants

M. K. ABBASI1, S. SHARIF1, M. KAZMI1, T. SULTAN2, & M. ASLAM2

1

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Department of Soil and Environmental Sciences, Faculty of Agriculture, University of Azad Jammu and Kashmir,
Rawalakot, Azad Jammu and Kashmir, Pakistan and 2Soil Biology and Biochemistry Section, National Agriculture Research
Center, Islamabad, Pakistan

Abstract
This study was conducted to isolate plant growth promoting rhizobacteria (PGPR) from wheat rhizosphere and to evaluate
their potential use for improving growth, yield and nutrient uptake of wheat. Eight PGPR strains were isolated and studied
for their morphological and cultural characteristics, phosphate solubilization and indole acetic acid (IAA) production. All
isolates produced IAA ranging from 5.531.0 mg/ml, while four isolates were P-solubilizers. On the basis of morphological
characteristics, IAA production and P solubilization, strains WPR-32, WPR-42, and WPR-51 were identified as PGPR and
selected for further study. Efficiency of these three PGPR isolates and their mixtures (combinations) at two N levels (N at the
rate of 50 and 100 kg ha71) was evaluated in wheat under greenhouse conditions. Application of PGPR significantly
increased plant height, shoot fresh weight and shoot dry weight by 25, 45, and 86%, respectively, while increase in root
length, root fresh and dry weight was 27, 102, and 76%, respectively, over the un-inoculated control. PGPR also increased
number of tillers per plant, 1000-grain weight and grain yield by 23, 48 and 59% over the control. Uptake of N and P by
plant shoot was increased by three-fold, while K uptake was increased by 58% with PGPR application. The rate of increase in
growth, yield and nutrient accumulation was even higher in treatments receiving combined application of PGPR and Nfertilizer. The concentration of NO
N and available P in soil also increased with PGPR treatments. Results indicated that
3
among the PGPR isolates, efficiency of mixture of isolates was higher than the individual isolates and isolate S7 was relatively
more consistent than the remaining strains. This study demonstrated that application of PGPR with N fertilizer increased the
fertilizer N efficiency by increasing N content and N uptake in plants. This study also indicated that use of PGPR had similar
effects on growth, yield and nutrient uptake of wheat when compared with the low N level, i.e. N50. Therefore, application of
PGPR with low fertilizer rates could be a viable supplementary strategy for maximum benefits in terms of cost of production
and sustaining productivity. However, the applicability of this approach has to be tested in further field studies.

Keywords: Biofertilizer, inoculation, nutrient uptake, plant growth promoting rhizobacteria (PGPR)

Introduction
Plant growth promoting rhizobacteria (PGPR) are
free-living soil-borne bacteria that aggressively colonize the rhizosphere/plant roots and when applied to
seed or crops enhance the growth and yield of plants
(Kloepper et al. 1980). Vessey (2003) explained
PGPR as a wide variety of soil bacteria which, when
grown in association with a host plant, result in
stimulation of growth of their host. These rhizosphere bacteria enhance plant growth and yield either
directly or indirectly. The direct mechanisms of plant

growth promotion may involve the synthesis of

substances by the bacterium or facilitation of the
uptake of nutrients from the environment (Glick
et al. 1999). The indirect promotion of plant growth
occurs when PGPR lessens or prevents the deleterious effects of plant pathogens on plants by production of inhibitory substances or by increasing the
natural resistance of the host (Cartieaux et al. 2003).
The direct growth promoting mechanisms are as
follows: (i) nitrogen fixation; (ii) solubilization of
phosphorus; (iii) sequestering of iron by production
of siderophores; (iv) production of phytohormones

Correspondence: M. K. Abbasi, Department of Soil and Environmental Sciences, Faculty of Agriculture, University of Azad Jammu and Kashmir, Rawalakot,
Azad Jammu and Kashmir, Pakistan. Tel: 92 0 58249 60046. Fax: 92 0 58710 42826. Email: kaleemabbasi@yahoo.com
ISSN 1126-3504 print/ISSN 1724-5575 online 2011 Societa` Botanica Italiana
DOI: 10.1080/11263504.2010.542318

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160

M. K. Abbasi et al.

such as auxins (indole acetic acid (IAA)), cytokinins,

gibberellins and (v) lowering of ethylene concentration (Kloepper et al. 1989; Glick et al. 1999).
Frankenberger and Arshad (1995) have discussed
in detail the role of auxins, cytokinins, gibberellins,
ethylene and absicisic acids in improving the growth
and yield of crops when applied to plants. The
indirect mechanisms of plant growth promotion by
PGPR include (i) antibiotic production; (ii) depletion of iron from the rhizosphere; (iii) synthesis of
antifungal metabolites; (iv) production of fungal cell
wall lysing enzymes; (v) competition for sites on
roots and (vi) induced systemic resistance (Glick
et al. 1999).
Several studies clearly demonstrated the positive
and beneficial effects of PGPR on growth and yield
of different crops at different climates, soils and
temperatures. Wu et al. (2005) reported that
microbial inoculum Bacillus megaterium and Bacillus
mucilaginous not only increased the plant growth but
also improved nutritional assimilation of plant (total
N, P, and K). Egamberdiyeva and Hoflich (2004)
reported that application of PGPR increased root
and shoot dry weight (SDW) of cotton to a
maximum of 32 and 38%, respectively, while the
correspondence increases in NPK uptake was 13,
39, and 10%, respectively, above the control. It has
also been reported that wheat yield increased up to
30% with Azotobacter inoculation and up to 43%
with Bacillus inoculation (Kloepper et al. 1991). A
series of laboratory experiments with rhizobial
inoculation of wheat conducted by Zahir et al.
(2004) also demonstrated increases in root elongation (up to 20%), root dry weight (up to 13%),
shoot elongation (up to 38%) and SDW (up to
36%). Seed inoculation of barley with different
PGPR increased root weight by 8.916.7% and
shoot weight by 28.634.7% over the control
(Canbolat et al. 2006). Shaharoona et al. (2008)
conducted an experiment on the effect of combined
use of PGPR and N fertilizer on wheat and reported
that strain P. fluorescens significantly increased the
root weight by 1943%, number of tillers per
plant 1021%, grain yield 1543% and straw yield
2239% over respective un-inoculated control,
respectively. The inoculation of maize with bacteria
strains Pseudomonas alcaligenes, Bacillus polymyxa,
and Mycobacterium phlei significantly increased the
SDW (1730%), root dry weight (1952%) and
total dry matter of maize increased up to 38%
(Egamberdiyeva 2007).
Keeping in view the beneficial effects of PGPR, a
lab and greenhouse study was conducted to isolate
the bacterial strains from wheat rhizosphere, identified them and evaluated their efficiency as PGPR on
growth performance and nutrient uptake of wheat in
pots under greenhouse conditions.

Materials and methods

Isolation and identification of bacteria
Rhizosphere soil samples were collected from wheat
(Triticum aesiivum L.) growing fields at the Research
farms, National Agriculture Research Centre, Islamabad Pakistan when the crop was 60 days old. For
this purpose, plants from the wheat field were
randomly uprooted with a scoop keeping the root
system and adhering soil intact. PGPR were isolated
from the soil on the roots and rhizosphere of already
collected plant samples. The serial dilutions for 1071
to 1076 were made in already autoclaved McCartney
vials. Ten grams of soil from each sample was
aseptically weighed, transferred to an Erlenmeyer flask
with 100 ml sterile water and shaken for 30 min at 150
rpm (Sharpley 1960). Immediately after shaking, a
series of 10-fold dilutions of the suspension was made
as described by Okon et al. (1977). Rhizobacteria
isolates were selected to represent distinct types based
on differences in colony morphology, including colony
form. Isolates were re-streaked on nutrient agar (NA)
and checked for purity.
Medium and purification of bacteria
Isolation of bacterial strains was carried out in an Nfree solid Okon Media III (Okon et al. 1977).
Colonies developed on each specified medium were
picked and purified by streaking on plates containing
same media for studying colony morphological and
biochemical characteristics. The purified strains were
cultured on solid media for the observation of
different colony morphological characteristics, e.g.
colony colour, colony margins, colony surface
texture, elevation and pigmentation associated with
colonies. All the morphological and cultural characteristics of the bacterial strains were studied by
light microscopy (Gopala 1967). The isolated and
purified strains were characterized for gram staining
(Vincent 1970) and slides were prepared and studied
for cell morphology characteristics, i.e. cell shape/
size, cell structure.
Screening of bacterial isolates isolated from wheat and
tested for IAA production
Eight different isolates were identified as PGPR by
staining, morphological, cultural, growth and biochemical characters (Table I). These isolates were
designated as WPR-42; WPR-50; WPR-51; WPR43; WPR-32; WPR-52; WPR-62 and WPR-63.
Isolates were further tested for indole-3-acetic acid
(IAA) production. For this purpose, cultures were
grown in Okons malate medium (Okon et al. 1977)
following Yasmin et al. (2004). Tryptophan (100 mg/
l) was added as the precursor of indole-3-acetic acid.

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PGPR and wheat growth

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Table I. Morphological, physiological, cultural characteristics and IAA production of PGPR bacterial strains isolated from wheat
rhizosphere.

PGPR
strains

Gram
stain

Shape of bacteria color

Colony color
on NA

WPR-32
WPR-42
WPR-51
WPR-43
WPR-32
WPR-52
WPR-62
WPR-63

7ve
ve
7ve
7ve
7ve
7ve
7ve
7ve

Slightly curved rods

Rod
Rod
Cocci-bacilli/oval
Slightly curved rods
Slightly curved rods
Rod
Slightly curved rods

Light pink
Off white
Light green
Off-white
Light pink
Dark pink
White
Light pink

After 1 week of growth, qualitative estimations of

indole-3-acetic acid were performed by Fe-HClO4
and Fe-H2SO4 reagents. The ethyl acetate oxidation
method was used for a quantitative estimation of
indole-3-acetic acid by HPLC using Turbochrom
software (Perkin Elmer, USA) (Hameed et al. 2004).
Phosphate solubilization
A single colony of bacterial culture grown on LuriaBertani medium was streaked onto Pikovskaias
medium containing tricalcium phosphate (Pikovskaia
1948) and incubated at 30 + 18C for 710 days. The
plates were observed for clear P-zone formation
around the colonies. Quantification of available
phosphorus solubilized by the bacterial isolate was
quantified by the phospho-molybdate blue colour
method (Gull et al. 2004). Fresh bacterial culture
was grown in Pikovskaia broth on a rotary shaker for
12 days at 24 + 18C. The suspension was centrifuged at 6000g for 15 min. The supernatant was
decanted and filtered, and the pH of the sample was
analyzed. The available phosphorus was determined
at 882 nm using a spectrophotometer and calibrated
with a standard phosphate curve.
Selection of isolates and plant infectivity test under
greenhouse conditions
On the basis of morphological characteristics and
IAA production potential, three potential strains of
PGPR, i.e. WPR-32, WPR-42 and WPR-51 were
identified and selected for invitro studies. A total of
seven PGPR strain treatments including three
selected strains and their possible combinations
(multiple strains, mixture of strains) including a
control were used to evaluate their effects on the
growth, yield and nutrient uptake of wheat. Pots
were used for this study having size of 150 in
diameter. Pots were sterilized with 20% sodium
hypochlorite solution, filled with 12 kg soil. The soil
used in the study was sandy loam, had an organic

Colony size/shape on NA

Phosphate
solubilization
ability

IAA produced
(mg/ml)

Irregular size with wrinkled surface

Regular size with crenate boarders
Irregular size with swarming growth
Regular size/circular
Irregular size (wrinkled surface)
Irregular size (entire edges)
Irregular size (rough surface)
Irregular size (wrinkled surface)

ve
ve
ve
7ve
7ve
7ve
ve
7ve

24.7
31.0
20.2
13.1
11.1
9.8
5.5
8.5

matter content 1.12%, total N 0.029%, available P

6.5 mg kg71 (Olsen & Sommers 1982), available K

125 mg kg71, NH
N contents 8.2
4 N and NO3 
71
and 3.6 mg kg , respectively, cation exchange
capacity 24.3 cmol kg71soil and pH 7.3. Soil
samples were sterilized by autoclaving at 1218C at
1.1 atm pressure in a Pyrex beaker for 2 h and then
cooled for 24 h.
Pots were arranged according to the following
treatments: (a) PGPR strains 08 including control,
i.e. control (without bacteria inoculation), WPR-32,
WPR-42, WPR-51, WPR-32 WPR-42, WPR32 WPR-51, WPR-42 WPR-51, WPR-32
WPR-42 WPR-51 designated as S0, S1, S2, S3, S4,
S5, S6, S7; (b) mineral N fertilizer 03 i.e. control
(N0, without N fertilizer), N at the rate of 50 kg ha71
(N50) and N at the rate of 100 kg ha71 (N100); (c)
replications 03. Basal dose of P and K was applied
in all pots at the rate of 80 and 70 kg P2O5 and K2O
ha71, respectively, in the form of single super
phosphate and sulphate of potash (K2SO4), respectively. Mineral N was applied in the form of urea. All
the fertilizers including N were applied and mixed
thoroughly in soil just before sowing.
Seeds of wheat var. GA-2002 were surface
sterilized in 70% ethanol for 2 minutes and in
1.2% sodium hypochlorite for 10 minutes and then
washed three times with sterile water. Seeds were
inoculated by immersion in the appropriate PGPR
suspensions, 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. Seedlings were
watered daily to maintain the moisture at approximately 60% water holding capacity of the soil.
Biological and chemical analyses
For growth study, two plants from each pot were
uprooted 50 days after sowing, with minimal damage
to the root system, washed gently under running tap
water to remove the adhering soil particles. The root

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162

M. K. Abbasi et al.

system was separated from the shoots, which were

oven dried for 3 days at 708C. Total root length was
measured as described by Farrell et al. (1993). Shoot
length (SL), shoot and root fresh weight and shoot
and root dry weight were also recorded. Remaining
plants were harvested at complete maturity at the end
of the study. The number of tillers per plant was
recorded before final harvest. Similarly, grain yield
per plant and 1000-grain weight of wheat were also
recorded. The harvested plants were rinsed with
deionized water and oven-dried at 758C for 72 h.
The dried shoot tissues were ground and then
digested using concentrated HNO3 for the determination of K using an atomic absorption spectrometer. Total nitrogen contents of the plants were
determined by the Kjeldahl method (Keeney &
Nelson 1982). Total P of plants was determined
colorimetrically by the molybdophosphate method
(Murphy & Riley 1962). Soil samples collected from
each treatment at the end and used for the
determination of available N (NO
N) and avail3
able-P analysis using the micro-Kjeldahl and sodium
bicarbonate extraction (Olsen & Sommers 1982),
respectively.
Statistical analysis
The data regarding the effect of inoculation with
PGPR at different fertilizer levels were analysed
using analysis of variance followed by statistical
software package Statgraphics (1992). Least significant differences are given to indicate significant
variations among the values of either PGPR or N
fertilizer or interaction between the two. 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

Identification and selection of plant growth promoting
rhizobacteria (PGPR)
In this study, eight rhizobacterial isolates were
isolated from wheat rhizosphere and identified as
PGPR on the basis of their morphological, physiological and cultural characteristics as well as their
ability to produce growth hormones and solubilized
insoluble phosphate. All strains except WPR-42 were
Gram-negative and fast-growing motile rods or oval
of different sizes. The colonies produced were
irregular or regular surfaces, circular, translucent,
opaque, white, off-white and yellow with smooth or
irregular margins (Table I). The variation in morphological characteristics of different isolated
observed during the study was similar to that

reported previously by Hafeez et al. (2006). All

isolates were able to produced IAA ranging from
5.531.0 mg/ml. Isolate WPR-42 produced the highest IAA (31.0 mg/ml), followed by WPR-32 (24.7 mg/
ml) and WPR-51 (20.2 mg/ml). IAA production for
the remaining strains was in the range of 5.5
13.1 mg/ml, indicating variation in IAA production.
Similar to our findings, few workers also have
reported that IAA production by microbial isolates
varied greatly within species and/or strains of the
same species (Mansour et al. 1994; Zahir et al.
2000). Differences in the production of IAA among
bacterial strains can be attributed to the various
biosynthetic pathways, location of the genes involved, regulatory sequences and the presence of
enzymes to convert active free IAA into conjugated
forms (Patten & Glick 1996). The production of IAA
by bacteria isolated from rhizosphere of different
crops was already reported by Cakmakci et al. (2007)
from wheat, Hameeda et al. (2006) from pearl millet,
Hafeez et al. (2006) from wheat, maize and rice, Dey
et al. (2004) from peanut, Yasmin et al. (2004) from
rice. Four out of eight isolates tested exhibited the
potential for P solubilization, another important
characteristic for inoculum development. This indicated the possession of mineral phosphate solubilization function by these strains. Hafeez et al. (2006)
found only one out of 17 tested strains isolated from
cereals rhizosphere to be able to solubilize P.
Generally, it has been found that the organisms
isolated from the rhizosphere of legumes have been
found to be more efficient in solubilizing phosphates
than those from the non-rhizosphere or from the root
zone of non-legumes (Gull et al. 2004; Hameed et al.
2004). But the proportion of P solubiliser found here
in the rhizosphere of wheat (non-legumes) was
satisfactory and encouraging. Isolation and characterization of these beneficial bacteria may be an
indication of the environmental and ecological
conditions of the sampled area having potential to
produce and synthesize PGPR.
Effect of PGPR and mineral N on the growth
characteristics of wheat
An overview of the effect of PGPR and N fertilizer on
the growth of wheat plants is presented in Figure 1,
while the detailed values are given in Tables II and
III. Values in column-1 show strains (S) effect while
values in row-1 indicate the N effect (N). The
remaining values exhibit interactive effect of N 6 S.
Application of PGPR significantly increased SL
ranging between 3443 cm compared with 31 cm
in the control, shoot fresh weight (SFW) from 4 g in
the control to 5.36.3 g in PGPR treatments and
SDW from 0.63 g in the control to 0.731.50 g in
PGPR treatments. By taking the average of 7 strains

PGPR and wheat growth

163

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Figure 1. An overview of the effect of different PGPRs alone or in combination with N fertilizers on the growth of wheat plants.

Table II. Effect of three different bacterial strains, i.e. plant growth promoting rhizobacteria (PGPR) alone and their combinations
(mixtures) at two levels of mineral N on shoot characteristics, i.e. shoot length (cm), shoot fresh weight (g) and shoot dry weight (g) of wheat
grown in pots under greenhouse conditions (average of three repeats).
Shoot length (cm) N-level
PGPR strains

N0

N50

31.0
44.0
S0
42.0
46.3
S1
S2
36.0
41.0
S3
38.0
44.0
S4
34.0
41.0
37.0
48.0
S5
S6
41.0
46.0
S7
43.0
50.0
Mean
37.8
45.0
LSD (0.05) for PGPR strains 5.71
LSD (0.05) for N levels 1.77
LSD (0.05) for interaction 5.01

Shoot fresh weight (g) N-level

N100

Mean

N0

45.7
53.0
56.0
50.0
55.0
60.0
58.0
64.7
55.3

40.2
47.1
44.3
44.0
43.3
48.3
48.3
52.6

4.0
6.0
5.3
5.3
6.0
5.9
5.8
6.3
5.55

N50

N100

Mean

7.8
11.7
7.8
8.3
13.7
9.3
9.0
17.3
10.4
9.3
19.0
11.2
8.3
14.6
9.6
9.7
19.1
11.6
9.0
18.0
10.9
10.0
20.0
12.1
8.9
16.7
LSD (0.05) for bacterial
strains 1.93
LSD (0.05) for N levels 1.18
LSD (0.05) for interaction 3.34

Shoot dry weight (g) N-level

N0

N50

N100

Mean

0.63
1.47
0.73
1.50
1.13
0.90
1.13
1.33
1.10

1.6
2.4
1.53
1.6
2.8
1.96
1.8
3.2
1.91
1.7
3.0
2.10
1.8
2.97
1.98
1.5
3.50
2.11
1.9
3.4
2.13
1.97
3.9
2.40
1.78
3.15
LSD (0.05) for bacterial
strains 0.23
LSD (0.05) for N levels 0.14
LSD (0.05) for interaction 0.39

S0 control (without bacteria inoculation); S1 WPR-32; S2 WPR-42; S3 WPR-51; S4 WPR-32 WPR-42; S5 WPR-32 WPR51; S6 WPR-42 WPR-51; S7 WPR-32 WPR-42 WPR-51, whereas N0 control without N fertilizer; N50 N at the rate of 50 kg
ha71; N100 N at the rate of 100 kg ha71.

and comparing the values to those recorded from

control, application of PGPR increased SL by 25%,
SFW by 45% and SDW by 86% over the uninoculated control. The relative increase in root
length, root fresh weight and root dry weight was 27,
102 and 76%, respectively. Among different strains,
mixture of strains, i.e. S7 showed the maximum
values for most of the characteristics. Zahir et al.
(2004) conducted a series of laboratory experiments
with rhizobial inoculation on wheat and reported
increases in root elongation up to 20%, root dry
weight up to 13%, shoot elongation 38% and SDW
36%. Similar rate of increase in wheat due to the
application of PGPR is also reported by Cakmakci

et al. (2007). Result indicated that the highest values

for most of the growth characteristics achieved by
PGPR application (alone) are statistically equivalent
or in some cases greater than the values obtained
from the application of lower rate of N, i.e. N50 while
the combine application of strains 6 N50 resulted in
significant increase in SL, root length, root fresh
weight and root dry weight compared with the values
obtained from application of highest rate of mineral
N (N100).
Yield and yield components of wheat also showed
similar trend that observed for growth parameters
(Table IV). Application of PGPR alone increased
number of tillers, 1000-grain weight and grain yield

164

M. K. Abbasi et al.

Table III. Effect of three different bacterial strains, i.e. plant growth promoting rhizobacteria (PGPR) alone and their combinations
(mixtures) at two levels of mineral N on root characteristics, i.e. root length (cm), root fresh weight (g) and root dry weight (g) of wheat
grown in pots under greenhouse conditions (average of three repeats).
Root length (cm) N-levels
PGPR strains

N0

N50

13.0
15.0
S0
S1
15.0
17.0
S2
12.0
16.0
16.0
21.0
S3
S4
15.0
20.0
S5
19.0
22.0
S6
19.0
21.0
20.0
24.0
S7
Mean
16.13
19.5
LSD (0.05) for PGPR strains 2.19

Root fresh weight (g) N-levels

N100

Mean

N0

17.0
18.3
21.0
23.0
23.0
25.0
24.0
27.0
22.3

15.0
16.8
16.3
20.0
19.3
22.0
21.3
23.7

0.30
0.60
0.40
0.90
0.53
0.60
0.42
0.80
0.57

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LSD (0.05) for N levels 3.49

LSD (0.05) for interaction 3.88

N50

N100

Mean

0.90
1.13
0.78
1.20
1.50
1.10
1.40
2.10
1.30
1.33
2.00
1.41
1.35
1.90
1.26
1.43
2.90
1.64
1.36
3.20
1.66
1.56
3.90
2.09
1.32
2.33
LSD (0.05) for PGPR
strains 0.20
LSD (0.05) for N levels 0.12
LSD (0.05) for interaction 0.35

Root dry weight (g) N-level

N0

N50

N100

Mean

0.20
0.37
0.40
0.32
0.50
0.43
0.50
0.48
0.20
0.45
0.74
0.46
0.30
0.47
1.0
0.59
0.30
0.39
0.60
0.43
0.24
0.40
1.0
0.55
0.43
0.52
0.80
0.58
0.50
0.60
1.90
1.00
0.334
0.454
0.867
LSD (0.05) for PGPR strains 0.033
LSD (0.05) for N levels 0.052
LSD (0.05) for interaction 0.147

S0 control (without bacteria inoculation); S1 WPR-32; S2 WPR-42; S3 WPR-51; S4 WPR-32 WPR-42; S5 WPR-32 WPR51; S6 WPR-42 WPR-51; S7 WPR-32 WPR-42 WPR-51, whereas N0 control without N fertilizer; N50 N at the rate of 50 kg
ha71; N100 N at the rate of 100 kg ha71.

Table IV. Effect of three different bacterial strains, i.e. plant growth promoting rhizobacteria (PGPR) alone and their combinations
(mixtures) at two levels of mineral N on yield and yield components, i.e. number of tillers per plant, 1000-grain weight and grain yield (g per
plant) of wheat grown in pots under greenhouse conditions (average of three repeats).
Number of tillers per plant N-level
PGPR strains

N0

N50

4.0
5.0
S0
S1
5.2
6.2
S2
4.2
7.1
S3
5.0
8.2
2.7
6.3
S4
S5
5.4
7.1
S6
5.6
6.4
6.2
8.3
S7
mean
4.6
6.6
LSD (0.05) for PGPR strains 1.23
LSD (0.05) for N levels 0.75
LSD (0.05) for interaction 2.12

N100

Mean

6.1
6.2
7.1
7.4
6.3
8.2
7.4
9.2
7.0

5.0
5.9
6.1
6.9
5.1
6.9
6.5
7.9

1000-grain weight (g) N-level

N0

N50

N100

Grain yield (g per plant) N-level

Mean

N0

21.1
32.8
38.2
30.7
25.6
38.4
39.1
34.4
25.1
38.2
43.3
35.5
27.4
40.5
44.0
37.3
31.3
39.9
46.2
39.1
35.6
43.3
46.7
41.9
35
44
48.2
42.4
38.4
44.7
50.0
44.5
29.9
40.2
44.5
LSD (0.05) for PGPR strains 3.19

2.3
2.8
3.1
3.7
3.5
4.1
3.9
4.5
3.5

LSD (0.05) for N levels 2.18

LSD (0.05) for interaction 4.34

N50

N100

Mean

3.7
4.3
3.4
4.4
4.4
3.9
4.4
4.8
4.1
4.6
4.8
4.4
4.6
5.0
4.4
4.8
5.3
4.7
5.1
5.6
4.9
5.6
5.9
5.3
4.7
5.0
LSD (0.05) for PGPR
strains 0.42
LSD (0.05) for N levels 0.34
LSD (0.05) for interaction 1.12

S0 control (without bacteria inoculation); S1 WPR-32; S2 WPR-42; S3 WPR-51; S4 WPR-32 WPR-42; S5 WPR-32 WPR51; S6 WPR-42 WPR-51; S7 WPR-32 WPR-42 WPR-51, whereas N0 control without N fertilizer; N50 N at the rate of 50 kg
ha71; N100 N at the rate of 100 kg ha71.

by 23, 48 and 59% (average), over the un-inoculated

control. Variation in the efficiency among different
strains was apparent and mixture of strains performed better than the individual strains. Shaharoona et al. (2008) recently reported that strain P.
fluorescens significantly increased the number of
tillers per plant 1021%, grain yield 1543% and
straw yield 2239%, over respective un-inoculated
controls. The maximum values for yield and yield
components in our case were obtained following the
combine application of PGPR with N50 and N100.

Grain yield in combine treatments of PGPR and N50

(S 6 N50) increased ranging between 4.45.6 g
compared with the grain yield of 3.7 g in N50 and
2.84.5 g in treatments receiving only PGPR.
Similar trend in yield was found in the combine
treatments of PGPR 6 N100.
Application of PGPR alone or in combination with
N fertilizer significantly increased N, P, and K
accumulation in wheat (Table V). For example,
application of PGPR alone increased N content in
wheat shoot from 1.2% in the un-inoculated to

PGPR and wheat growth

165

Table V. Effect of three different bacterial strains, i.e. plant growth promoting rhizobacteria (PGPR) alone and their combinations (mixtures)
at two levels of mineral N on N content and N uptake of wheat shoot and root grown in pots under greenhouse conditions (average of three
repeats).
N content in shoot (%)
N-level
PGPR strains
S0
S1
S2
S3
S4
S5
S6
S7
Mean
LSD (0.05) for

N0
1.2
1.7
1.8
1.6
1.46
1.7
1.9
2.43
1.71
PGPR

N50

N100

2.23 2.46
2.36 2.60
2.46 2.20
2.49 2.52
2.43 2.59
2.8
2.78
2.48 2.63
2.91 2.89
2.52 2.58
strains 0.21

LSD (0.05) for N levels 0.146

Downloaded By: [University of Sussex] At: 11:39 5 March 2011

LSD (0.05) for interaction 0.312

Mean
1.96
2.18
2.15
2.20
2.16
2.43
2.34
2.74

Plant N uptake (mg per plant)

N-level
N0

N50

N100

Mean

7.6 35.7
59.0
34.1
23.1 37.8
72.8
44.5
13.1 44.3
70.4
42.6
24.0 42.3
75.6
47.3
16.5 43.7
76.9
45.7
15.3 42.0
97.3
51.5
21.5 47.1
89.4
52.7
32.3 57.3 112.7
67.5
19.2 43.8
81.86
LSD (0.05) for PGPR
strains 5.23
LSD (0.05) for N
levels 6.72
LSD (0.05) for
interaction 9.14

N content in root (%)

N-level
N0

N50

N100

Mean

1.17 0.95 1.07

1.06
1.65 1.44 1.14
1.41
1.69 1.35 1.27
1.44
1.59 1.66 1.63
1.63
1.57 1.40 1.70
1.56
1.6
1.67 1.97
1.75
1.58 1.60 1.07
1.42
1.78 1.78 2.09
1.88
1.58 1.48 1.49
LSD (0.05) for PGPR
strains 0.095
LSD (0.05) for N
levels 0.058
LSD (0.05) for
interaction 0.164

Root N uptake (mg per plant)

N-level
N0

N50

N100

Mean

2.3
3.5
4.3
3.38
8.3
6.2
5.7
6.71
3.4
6.1
9.4
6.28
4.8
7.8
16.3
9.62
4.7
5.5
10.2
6.79
3.8
6.7
19.7
10.07
6.8
8.3
8.6
7.89
8.9
10.7
39.7
19.76
5.37
6.84 14.23
LSD (0.05) for PGPR
strains 1.19
LSD (0.05) for N
levels 0.34
LSD (0.05) for
interaction 1.12

S0 control (without bacteria inoculation); S1 WPR-32; S2 WPR-42; S3 WPR-51; S4 WPR-32 WPR-42; S5 WPR-32 WPR51; S6 WPR-42 WPR-51; S7 WPR-32 WPR-42 WPR-51, whereas N0 control without N fertilizer; N50 N at the rate of 50 kg
ha71; N100 N at the rate of 100 kg ha71.

1.702.43% with an average value of 1.80% indicating 50% increase in N contents due to PGPR
application. The increase in N-uptake was even
higher and over three-fold increase in N-uptake due
to PGPR was observed. However, combined application of PGPR with N resulted in the highest
N-uptake and the maximum N-uptake of 57 mg
plant71 and 113 mg plant71 was recorded in S7N50
and S7N100, respectively. Zaidi and Khan (2005)
found 2794% increase in N uptake by wheat shoot
following the application of different strains of
PGPR. Likewise, inoculants caused 50% increase
in P content of plant shoot and P uptake was
increased by three-fold (Table VI). An increase in Puptake over control may be due to the bacterial
solubilisation of insoluble phosphates in the soil. The
rhizosphere of cereal crops was found to be a harbor
of great number of phosphate solubilizing bacteria
(Narula et al. 2000) and four out of eight tested
isolates were found P-solubilizer in this study. These
bacteria showed an effective role in P uptake and
growth promotion of plants by dissolution of
inorganic insoluble phosphate. Bacteria isolated from
the rhizosphere are capable of increasing availability
of phosphorus to plants either by mineralization of
organic phosphate or by solubilization of inorganic
phosphate by production of acids (Lifshitz et al.
1987). Adesemoye et al. (2008) reported similar
findings in maize grown under field conditions.
Potassium concentration in plants under different
treatments ranged from 1.15 (control) to 1.36%
(average of 07 inoculants) (Table VI) showing 18%

increase while the uptake of K due to PGPR was

2-fold over the control. Sheng and He (2006)
explained that organic acids, e.g. citric, oxalic,
tartaric, succinic, etc. produced by the PGPR are
able to chelate metals and mobilize K from K-containing minerals. Increased nutrient uptake by plants
inoculated with effective strains has been attributed
to the production of plant growth regulators by the
bacteria at the root interface, which stimulated root
development and resulted in better absorption of
water and nutrients from the soil as reported by
Lifshitz et al. (1987). In this study, IAA was
produced by all the tested PGPR strains that
contributed to increase root development thereby
increased nutrient uptake. PGPR are reported to
enhance NPK uptake in different crops, i.e. in cotton
and pea (Egamberdiyeva & Hoflich 2004); in wheat
(Khan & Zaidi 2007) and in maize (Wu et al. 2005;
Egamberdiyeva 2007).
Soil analysis (after crop harvest) indicated that soil
NO
N and P content were significantly increased
3
by PGPR and mineral N fertilizer (Table VII). The
increased N content may have been due to the
increase in different fractions of mineral N in soil or
increase in plant growth stimulating mechanisms as
result of N2 fixation, which can positively influence
plant growth and crop yields (Asghar et al. 2004). In
addition, another important characteristic of PGPR
associated with increase in soil N is excretion of
ammonia in the rhizosphere in the presence of root
exudates (Narula & Gupta 1986), which could
explain the reasons for high NO
N in inoculated
3

166

M. K. Abbasi et al.

pots. Wu et al. (2005) also found increase in N and P

concentration in soil after PGPR application. The
increase in soil P content might be due to the Psolubilizing potential of two isolates and their
mixtures used in the experiment. Wu et al. (2005)
also found increase in N and P concentration in soil
after PGPR application.
This study demonstrated that application of PGPR
with mineral N may increase the N use efficiency of
applied N, i.e. N50 and N100. For example, N uptake
in the treatments receiving sole application of N

fertilizer (N50 and N100 in un-inoculated control)

was 36 and 59 mg per plant, respectively. Application of strains alone or their mixture with N fertilizer
increased N uptake ranging between 3857 mg per
plant in N50 and 70113 mg per plant in N100,
showing almost two-fold increase in N uptake.
Another important finding was the growth, yield
and nutrient uptake in wheat under seven PGPR
treatments (without N fertilizer) were almost equal
or comparable to those found in the lower level of N,
i.e. N50. For example, average grain yield of 07

Table VI. Effect of three different bacterial strains, i.e. plant growth promoting rhizobacteria (PGPR) alone and their combinations
(mixtures) at two N levels on P content, P uptake, K content and K uptake of wheat grown in pots under greenhouse conditions (average of
three repeats).

Downloaded By: [University of Sussex] At: 11:39 5 March 2011

P content in shoot (%)

N-level
PGPR strains
S0
S1
S2
S3
S4
S5
S6
S7
Mean
LSD (0.05) for

N0
0.18
0.24
0.24
0.21
0.30
0.28
0.28
0.34
0.26
PGPR

N50

N100

Mean

0.21 0.23
0.29 0.28
0.31 0.34
0.26 0.31
0.35 0.37
0.32 0.36
0.34 0.36
0.39 0.44
0.31 0.34
strains 0.02

0.21
0.27
0.30
0.26
0.34
0.32
0.33
0.39

LSD (0.05) for N levels 0.034

LSD (0.05) for interaction 0.061

P uptake (mg per plant)

N-level
N0

N50

N100

Mean

1.1
3.4
5.5
3.34
3.5
4.6
7.8
5.34
1.8
5.6
10.9
6.07
3.2
4.4
9.3
5.62
3.4
6.3
11.0
6.89
2.5
4.8
12.6
6.64
3.2
6.5
12.2
7.29
4.5
7.7
17.2
9.79
2.90 5.41 10.82
LSD (0.05) for PGPR
strains 0.62
LSD (0.05) for N
levels 1.18
LSD (0.05) for
interaction 0.74

K content in shoot (%)

N-level
N0

N50

N100

Mean

1.15 1.23 1.32

1.23
1.27 1.39 1.41
1.36
1.23 1.32 1.40
1.32
1.31 1.41 1.49
1.40
1.41 1.48 1.61
1.50
1.33 1.43 1.56
1.44
1.39 1.53 1.65
1.52
1.57 1.70 1.72
1.66
1.33 1.44 1.52
LSD (0.05) for PGPR
strains 0.072
LSD (0.05) for N
levels 0.041
LSD (0.05) for
interaction 0.113

K uptake (mg per plant)

N-level
N0

N50

N100

Mean

7.2
19.7
31.7
19.54
18.7
22.2
39.5
26.80
9.0
23.8
44.8
25.85
19.7
24.0
44.7
29.44
15.9
26.6
47.8
30.13
12.0
21.5
54.6
29.34
15.7
29.1
56.1
33.63
20.9
33.5
67.1
40.48
14.88 25.04 48.28
LSD (0.05) for PGPR
strains 2.13
LSD (0.05) for N levels 2.34
LSD (0.05) for
interaction 3.12

S0 control (without bacteria inoculation), S1 WPR-32, S2 WPR-42, S3 WPR-51, S4 WPR-32 WPR-42, S5 WPR-32 WPR51, S6 WPR-42 WPR-51, S7 WPR-32 WPR-42 WPR-51, whereas N0 control without N fertilizer; N50 N at the rate of 50 kg
ha71; N100 N at the rate of 100 kg ha71

Table VII. Effect of application of plant growth promoting rhizobacteria (PGPR) alone and their combinations (mixtures) at two N levels on
71
the changes in NO
) and available P (mg kg71) in soil (average of three repeats).
3  N (mg kg
71
) N-level
NO
3 N (mg kg

PGPR strains

N0

6.4
S0
9.8
S1
S2
7.2
S3
10.0
S4
10.1
9.2
S5
S6
10.5
S7
11.7
Mean
9.4
LSD (0.05) for PGPR strains 1.23
LSD (0.05) for N levels 2.02
LSD (0.05) for interaction 2.23

Available P (mg kg71) N-level

N50

N100

Mean

N0

10.4
12.3
12.1
12.6
14.0
11.8
13.2
13.7
12.5

12.9
15.0
13.4
15.2
17.6
17.0
16.5
20.7
16.0

9.9
12.4
10.9
12.6
13.9
12.7
13.4
15.4

6.7
9.2
6.9
10.9
9.7
10.2
10.0
11.0
9.3

N50

N100

6.9
7.1
9.8
10.3
8.5
9.24
11.3
12.4
10.9
10.1
11.8
12.0
10.0
12.3
12.2
12.4
10.2
10.7
LSD (0.05) for PGPR strains 1.02
LSD (0.05) for N levels 1.12
LSD (0.05) for interaction 3.12

Mean
6.9
9.8
8.2
11.6
10.2
11.3
10.7
11.9

S0 control (without bacteria inoculation); S1 WPR-32; S2 WPR-42; S3 WPR-51; S4 WPR-32 WPR-42; S5 WPR-32 WPR51; S6 WPR-42 WPR-51; S7 WPR-32 WPR-42 WPR-51, whereas N0 control without N fertilizer; N50 N at the rate of 50 kg
ha71; N100 N at the rate of 100 kg ha71.

Downloaded By: [University of Sussex] At: 11:39 5 March 2011

PGPR and wheat growth

PGRP treatments (without N fertilizer) was 3.66 mg
per plant in comparison with the yield of 3.70 mg per
plant at N50. Similarly, the maximum uptake of N, P,
and K by plants in the PGPR treatments (average)
was 32.3, 4.5, and 20.9 mg per plant observed at S7
(mixture of all the three strains) while the N, P, and K
uptake at lower level of N fertilizer (N50) was 35.7,
3.4, and 19.7 mg per plant, respectively. These
findings demonstrated that application of PGPR to
soil could compensate for nutrient deficiency and
maintain, at least partly, the immediate need of
nutrient requirement of plants. The PGPR therefore
may have a potential to decrease the input cost of
agricultural production and can replace the use of
almost 50% of the mineral N fertilizer if applied with
50% of the required N. In addition, the use of PGPR
with low fertilizer rate is also an environment friendly
step and would be a viable supplementary strategy for
further increasing crop yields. It could be concluded
that PGPR technology should be employed with
appropriate doses of fertilizers to get maximum
benefit in terms of fertilizer savings and better growth
any yield of crops. However, the real recommendation can be suggested after conducting long duration
experiments under field conditions. Results indicated
that among the PGPR isolates, the mixture of all
strains, i.e. S7 was relatively more consistent in
improving the different growth parameters of wheat,
which may imply that combining different strains and
using their mixtures instead of individual strain alone
could be a useful technique for better growth and
productivity of crops. From these results, we can
conclude that rhizosphere of different plants and
crops are the original harbour of important and
beneficial bacteria. These growth promoting bacteria
can be isolated from the soil of different crops root
zones and used for enhancing crop growth/yield and
improving soil fertility.

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