Archives of Agronomy and Soil Science, 2015

Vol. 61, No. 12, 1691–1705, http://dx.doi.org/10.1080/03650340.2015.1036044

Halotolerant rhizobacteria: beneficial plant metabolites

and growth enhancement of Triticum aestivum L. in
salt-amended soils
Asif Raheem and Basharat Ali*

Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore, Pakistan
(Received 27 November 2014; accepted 25 March 2015)

Salt-tolerant strains of Enterobacter asburiae, Bacillus thuringiensis, Moraxella

pluranimalium and Pseudomonas stutzeri were evaluated for their ability to alleviate
salt stress of wheat (Triticum aestivum L.) seedlings. 1-Aminocyclopropane-1-carbox-
ylate deaminase activity of P. stutzeri S-80 and B. thuringiensis S-26 was 190 and
183 nmol h−1, respectively. Maximum levels of auxin were recorded with P. stutzeri S-
80 (107 µg ml−1) and E. asburiae S-24 (143 µg ml−1) under normal and salt-stressed
conditions (0.25M NaCl), respectively, with 500 µg ml−1 L-tryptophan. Auxin
response mediated by rhizobacteria was also demonstrated by microscopically assay-
ing the transgenic auxin-responsive reporter DR5::GUS expression tomato (Solanum
lycopersicum L. cv. MicroTom). In pot trials, seedlings fresh and dry biomass wit-
nessed highly significant improvements of 1- and 2.2-folds, respectively, with M.
pluranimalium S-29 (at 100 mM NaCl) and E. asburiae S-24 (150 mM NaCl), over
control. At final harvest, maximum increase in number of tillers (up to 94%) and seed
weight (up to 40%) was recorded with E. asburiae S-24 and M. pluranimalium S-29 at
200 mM salt stress. In conclusion, newly isolated strains of M. pluranimalium S-29, E.
asburiae S-24 and P. stutzeri S-80 enhanced the growth of T. aestivum by mitigating
the salt stress of plants.
Keywords: halotolerant rhizobacteria; ACC-deaminase; bacterial auxin production;
salt-stress alleviation; plant growth promoting rhizobacteria

Introduction
Salt accumulation in soils is one of the major causes of declining crop productivity in
many arid and semi-arid regions of the world. Salinity affects virtually all aspects of a
plant’s physiology by changing the cellular water and ionic status. Plant performance
usually expressed as a crop yield, plant biomass or crop quality may be adversely affected
by salinity-induced nutritional disorders. These disorders may result from the effect of
salinity on nutrient availability, competitive uptake, transport or partitioning within the
plant (Setia et al. 2013; Shafi et al. 2013). In the present scenario, biotechnological
strategies based on the ability of crops and microbes to grow under abiotic stresses can
be an effective approach for sustainable agriculture. A number of studies have been
published regarding the activity of some plant growth-promoting rhizobacteria (PGPR)
to confer upon plants the ability to migitate abiotic stresses such as salt, drought and
nutrient deprivation (Grichko & Glick 2001; Mayak et al. 2004; Yang et al. 2009;

*Corresponding author. Email: basharat.ali.mmg@pu.edu.pk

© 2015 Taylor & Francis

1692 A. Raheem and B. Ali

Timmusk et al. 2011; Dodd & Pérez-Alfocea 2012). PGPR employ several direct mechan-
isms to enhance plant growth and yield. Auxin production and 1-aminocyclopropane-1-
carboxylate (ACC) deaminase are well documented in literature (Compant et al. 2005;
Onofre-Lemus et al. 2009; Ali et al. 2009a; Taghavi et al. 2010).
Indole-3-acetic acid (IAA) is the most abundant type of auxin produced by rhizobac-
teria, and its effects on plants are similar to that of exogenous IAA. PGPR that produce
IAA enhanced the growth and yield of agronomically important crops with a direct linear
positive correlation between in vitro auxin production and plant growth promotion by
PGPR (Khalid et al. 2004; Ali et al. 2009a, 2009b; Akhtar & Ali 2011). In various studies,
it has been suggested that increased root proliferation is related to microbial IAA
biosynthesis (Patten & Glick 2002). The precursor required for microbial IAA biosynth-
esis has been identified as L-tryptophan (Patten & Glick 1996). Tryptophan in the rhizo-
sphere may originate from root exudates and release from microbial cell and damaged
roots (Khalid et al. 2004; Rothballer et al. 2005; Spaepen et al. 2007). IAA promotes
better bacterial adaptation to stress conditions, leading to improved survival and persis-
tence in the environment (Bianco & Defez 2009; Hu et al. 2010). Bacterial phytohor-
mones have been shown to alleviate salt stress of plants grown under conditions of soil
salinity (Egamberdieva 2009). Under abiotic stresses, a high level of ethylene produced by
plant is another factor that affects root growth and development. Deleterious effects of
ethylene can be reduced by PGPR that contain ACC-deaminase activity (Cheng et al.
2007). In recent years, rhizobacteria exhibiting ACC-deaminase activity has been used to
alleviate the deleterious effects of ethylene under stressed conditions of high salinity to
enhance crop growth and productivity (Zahir et al. 2009; Nadeem et al. 2010; Ahmad
et al. 2013; Qin et al. 2014).
Soil salinity is also responsible for low crop production in Pakistan. It is estimated that
about 6.30 million hectares of the total land available (79.6 Mha) are salt affected.
Therefore, the management of salt-affected soils is essential to meet an ever increasing
food demand. Bacteria colonizing the roots of plants growing in saline areas can act as
potential inoculants for the crop productivity management. Within this context, haloto-
lerant microbes that belong to different bacterial genera were isolated from the rhizosphere
of Acacia arabica (Lam.) Willd. growing in Pothohar, salt range of Pakistan. Pothohar
forms a part of the Sub-Himalayan mountains, which stretches around 180 km East–West
between the Jhelum and Indus rivers (Ghazi et al. 2012). The reason for the selection of
this place is that Pothohar has arid conditions all around the year with less rainfall that
increases the osmotic stress of plants. Rhizobacteria were screened for in vitro auxin
production and ACC-deaminase activity. Auxin response in the presence of rhizobacteria
was further examined by assaying a transgenic auxin-responsive reporter DR5::GUS in
MT-tomato (Solanum lycopersicum L. cv MicroTom). After in vitro screening, rhizobac-
teria were evaluated for their ability to alleviate the salt stress and to enhance the growth
of wheat (Triticum aestivum L). This study is the first to report the potential agricultural
significance of Enterobacter asburiae, Moraxella pluranimalium and Pseudomonas stut-
zeri to mitigate the salt stress of wheat.

Materials and methods

Isolation of bacterial strains
Soil samples were collected from the rhizosphere of Acacia arabica (Lam.) Willd. growing
in the semi-arid zone of Pothohar, salt range of Pakistan. One gram of rhizospheric soil was
 Archives of Agronomy and Soil Science 1693

thoroughly mixed in 99 ml of autoclaved distilled water to make 10–2, 10–4 and 10–6
dilutions. A 50 µl of each dilution was plated on L-agar supplemented with 0.5M, 0.1M,
1.5M and 2M NaCl. Plates were incubated at 37°C for 24 h. After incubation, bacterial
colonies showing prolific growth were picked and purified by many rounds of streaking.
Finally, five strains showing beneficial metabolic properties for plants were selected.
Halotolerance of rhizobacteria was also evaluated by growing strains in L-broth supple-
mented with NaCl concentrations as mentioned above. Strains were grown at 37°C by
incubating them on shaker for 24 h. After incubation, optical densities (600 nm) of the
cultures were recorded to observe the effect of NaCl on bacterial growth.

16S rRNA gene sequencing

For 16S rRNA gene sequencing, genomic DNA was extracted from overnight grown bacterial
cultures by using Genomic DNA Purification Kit (Promega, Madison, WI, USA). About
1 × 5-kb DNA fragment containing 16S rRNA gene was amplified using forward 27f
(5′-AGAGTTTGATCCTGGCTCAG-3′) and reverse primer 1522r
(5′-AAGGAGGTGATCCA(AG)CCGCA-3′) (Johnson 1994). Polymerase chain reaction
(PCR) amplification was performed by using 50 µl of Dream Taq™ Green PCR Master
Mix (Fermentas, Burlington, ON, Canada) with 0.5 µg of chromosomal DNA template and
0.5 µM of each primer. The reaction mixtures were incubated in a thermocycler Primus 96
(PeQLab, Erlangen, Germany) at 94°C for 5 min and passed through 30 cycles: denaturation
for 20 s at 94°C, primer annealing for 20 s at 50°C and extension at 72°C for 2 min. Final
extension was carried out at 72°C for 5 min. The amplified products were purified using
QIAquick Gel Extraction Kit (QIAGEN) and sequenced using 27f and 1522r primers by
sending to Eurofins (Lancaster, UK).

Bacterial auxin production

Auxin production was estimated by growing strains in 50 ml L-broth medium supple-
mented with 100, 200, 300, 400 and 500 µg ml−1 L-tryptophan. Auxin production was
also quantified in the presence of 0.25M, 0.5M, 0.75M and 1M NaCl that were also
amended with 500 µg ml−1 L-tryptophan. Flasks were inoculated with 100 μl of bacterial
cell suspension adjusted to 107 CFU ml−1. All flasks were incubated at 37°C for 72 h at
120 rpm in triplicate. After incubation, cells were removed from the culture medium by
centrifugation at 2300 × g for 15 min, and auxin was detected in 1 ml of supernatant using
Salkowski reagent (Tang & Borner 1979). A standard curve was constructed by using
different concentrations of standard IAA (Sigma-Aldrich, St. Louis, MO, USA) to deter-
mine bacterial auxin in culture supernatant.

ACC-deaminase activity
Bacterial isolates were screened for ACC-deaminase activity following the method
described by Li et al. (2011) with slight modifications. Bacterial strains were inoculated
into 20 ml L-broth and incubated overnight at 37°C for 24 h on an orbital shaker. After
incubation, 2 ml of each bacterial culture was harvested by centrifugation at 8000 × g for
5 min. Supernatant was discarded and the pellet was washed twice with 1 ml of liquid
Dworkin and Foster (DF) medium. Afterwards, pellet was suspended into 2 ml of DF
medium supplemented with 3 mM ACC substrate and incubated at 37°C on shaker for
2 h. For comparison, 2 ml of DF-ACC medium was also incubated as a control. After
1694 A. Raheem and B. Ali

incubation, 1 ml of each culture was centrifuged and 100 µl of supernatant was diluted to
1 ml with DF medium. An aliquot (60 µl) of each diluted supernatant was then used for
the 96-well PCR-plate ninhydrin-ACC assay and mixed with 120 µl of ninhydrin reagent.
The PCR plate was then heated on boiling water bath for 30 min. DF medium was used as
a blank, and each sample was run in triplicate. After development of Ruhemann’s purple
colour, 100 µl was transferred in a microtitre plate to measure the absorbance at 570 nm
by using microplate spectrophotometer (Epoch, BioTek, Winooski, VT, USA).

Root growth assays

Biological activity of bacterial auxin was demonstrated by performing bioassays with
tomato (S. lycopersicum L. cv. MicroTom) and wheat (T. aestivum L. cv. FD-2008).
Tomato seeds were obtained from Moles Seed Ltd. (Colchester, Essex, UK) and surface
sterilized with 0.5% sodium hypochlorite for 5 min. Similarly, wheat seeds obtained from
Punjab Seed Corporation (Lahore, Pakistan) were sterilized as mentioned above. Seeds
were washed three times with autoclaved distilled water. Five sterilized seeds were
aseptically transferred to petri plates containing autoclaved moistened double-filter
paper. For seed inoculation, strains were grown in L-broth medium and harvested by
centrifugation as mentioned above. Then cultures were re-suspended in sterilized distilled
water and adjusted to 107 CFU ml−1. For each plate, 4 ml of bacterial suspension was
applied and incubated in dark for 5 days at room temperature. Water-treated seeds were
used as control. After incubation, root and shoot lengths for all treatments were recorded.

GUS histochemical analysis

Auxin response in the presence of rhizobacteria was further examined by assaying a
transgenic auxin-responsive reporter DR5::GUS in MT-tomato. Seeds of MT-DR5::GUS
were surface sterilized and transferred in petri plate containing sterilized moistened filter
paper treated with 5 ml bacterial suspension (107 CFU ml−1). Five pre-germinated seeds
were incubated in petri plates for 5 days in dark. For GUS staining, seedlings were
incubated in 1 ml solution containing 10 mM sodium phosphate buffer (pH 7.2), 10 mM
ethylenediaminetetraacetic acid, 1 mM 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc)
and 1% (v/v) Triton X-100. To aid infiltration, the seedlings were subjected to vacuum for
30 min and then incubated at 37°C in the dark for 16 h. The samples were cleared
overnight in formaldehyde:alcohol:acetic acid (2:10:1) solution and observed for auxin
localization by using Olympus BX41 compound microscope fixed with a digital camera
(Olympus C-7070).

Pot trials under salt stress

Experiments were also performed to evaluate the potential of bacterial strains to alleviate
salt stress of wheat by growing plants in larger earthen pots (30 × 30 cm) filled with 10 kg
loamy soil. The soil had a pH 7.2, electrical conductivity 0.45 dS m−1 and 0.65% organic
matter. Seeds were sterilized as mentioned earlier and treated with bacterial cell suspen-
sion (107 CFU ml−1) for 25 min. Initially, 15 seeds were sown in each pot with six
replications, and after germination 10 uniform seedlings were maintained. After 3 and 6
weeks of germination, plants were subjected to salt stress by applying 0 mM, 100 mM,
150 mM and 200 mM solutions of NaCl to the field capacity of soil. Water and respective
salt-amended controls were also kept for comparison. Pots were arranged in completely
 Archives of Agronomy and Soil Science 1695

randomized design in the wire house of Department of Microbiology and Molecular

Genetics, University of the Punjab, under natural environmental conditions. Ten weeks
after germination, five plants were removed to record shoot length and fresh and dry
biomass of seedlings. At final harvest, vegetative and yield parameters of wheat were also
recorded.

Sodium content measurements

Sodium content of plants grown in the presence of different salinity levels as mentioned
above was measured by atomic absorption spectroscopy. For the quantification of sodium,
1 g of dried plant material (leaves) grown till maturity was ground and digested in 20 ml
of 1M nitric acid (HNO3) as described in Ali et al. (2014). Then, material was incubated at
70°C for 4 h and cooled at room temperature for 1 h. Afterwards, the content of tube was
centrifuged at 10,000 × g for 10 min. An aliquot of 500 µl of the supernatant was diluted
to 5 ml with Milli-Q water. The NaCl concentrations were measured using graphite
furnace atomic absorption spectroscopy (Hitachi-Science & Technology-Z-5000
Polarized Zeeman Flame/Graphite Furnace Atomic Absorption Spectrophotometer;
Schaumburg, IL, USA). The sodium concentration in plant material was calculated by
using standard curve that was constructed by using 1 to 100 mg NaCl.

Statistical analysis
Data for in vitro screening, bioassays and plant growth parameters were subjected to
analysis of variance using SPSS 20 program and means separated using Duncan’s multi-
ple range test (P ˂ 0.05). Correlation coefficients between bacterial metabolic properties,
salinity and plant growth parameters were also recorded (P ˂ 0.01 or P ˂ 0.05).

Results
16S rRNA gene sequencing
The sequences of 16S rRNA gene were analysed by comparison with sequences in
GenBank through BLAST (http://www.ncbi.nlm.nih.gov/BLAST). After comparison,
strains S-24, S-26, S-29, S-50 and S-80 showed 99%, 99%, 99%, 97% and 98% similarity,
respectively, with Enterobater asburiae, Bacillus thuringiensis, M. pluranimalium,
B. thuringiensis and P. stutzeri. The sequences have been deposited in the GenBank
under accession numbers KJ011875-KJ011879.

Halophility assay
For halophility assays, bacterial strains were grown in 0.5M, 1M, 1.5M and 2M NaCl.
Results indicated that most of the bacterial strains exhibited tolerance up to 1M salt
concentration (Figure 1). Overall, B. thuringiensis S-50 and P. stutzeri S-80 were the most
effective at resisting high medium salt content evident by comparing growth against the
respective controls. Nevertheless, increasing salt concentrations significantly decreased
the growth of E. asburiae (r = −0.953; P ˂ 0.05), B. thuringiensis (r = −0.916; P ˂ 0.05)
and M. pluranimalium (r = −0.899; P ˂ 0.05).
1696 A. Raheem and B. Ali

0M 0.5M 1M 1.5M 2M

2.5
b
Culture density (600 nm)

2
ab
c c c c c
1.5 bc c bc
bc a a b b a
1 a a
b a a
0.5 a a
a a
0
S-24 S-26 S-29 S-50 S-80
Strains

Figure 1. Halophility assay demonstrating the effect of different salt concentrations on the growth
of halotolerant rhizobacteria. Different letters on bars indicate significant difference between treat-
ments using Duncan’s multiple range test (P ˂ 0.05). Affiliations: S-24, E. asburiae; S-26, B.
thuringiensis; S-29, M. pluranimalium; S-50, B. thuringiensis; S-80, P. stutzeri.

Bacterial metabolic properties

Auxin production was determined in bacterial culture grown in the presence of different
L-tryptophan concentrations. Generally, increasing L-tryptophan concentrations enhanced
auxin production several folds as compared to unamended controls (Figure 2). It is evident
from highly significant positive correlation between in vitro bacterial auxin production
and L-tryptophan concentrations. Highly significant positive correlation was observed for
E. asburiae S-24 (r = 0.991; P ˂ 0.01) and M. pluranimalium S-29 (r = 0.984; P ˂ 0.01)
and P. stutzeri S-80 (r = 0.985; P ˂ 0.05). Overall, maximum auxin levels were recorded
with P. stutzeri S-80 (107 µg ml−1) and E. asburiae S-24 (92 µg ml−1) at 500 µg ml−1 L-
tryptophan. On the other hand, amendments of L-broth with lower NaCl concentrations
stimulated auxin production. However, increasing salt concentrations from 0.75 to 1M
decreased bacterial auxin production (Figure 3). Nevertheless, lower NaCl amendment
(0.5M) stimulated auxin production for E. asburiae S-24 (143 µg ml−1), M. pluranima-
lium S-29 (109 µg ml−1) and B. thuringiensis S-26 (104 µg ml−1). For ACC-deaminase
activity, P. stutzeri S-80, B. thuringiensis S-26, M. pluranimalium S-29, B. thuringiensis

S-24; r = 0.991** S-26; r = 0.873 S-29; r = 0.984** S-50; r = 0.864 S-80; 0.985*

120
Auxin (µg ml–I)

80

40

0
0 100 200 300 400 500
L-Tryptophan (µg ml–I)

Figure 2. L-tryptophan dependent auxin production by halotolerant rhizobacteria. The results

shown are representative of two repetitions of the experiment. Bars at different points indicate SE
for each treatment. Value ‘r’ indicates highly significant positive correlation between L-tryptophan
and bacterial auxin production. ** (P ˂ 0.01), * (P ˂ 0.05).
 Archives of Agronomy and Soil Science 1697

S-24; r = –0.959* S-26; r = –0.889 S-29; r = –0.880 S-50; r = –0.986* S-80; –0.992**

160

Auxin (µg ml–I)

120

80

40

0
0M 0.25M 0.5M 0.75M 1M
NaCl

Figure 3. Effect of different salt concentrations on bacterial auxin production at 500 µg ml−1 L-
tryptophan. The results shown are representative of two repetitions of the experiment. Bars at
different points indicate SE for each treatment. Value ‘r’ indicates highly significant negative
correlation between increasing salt concentrations and bacterial auxin production. ** (P ˂ 0.01),
* (P ˂ 0.05).

S-50 and E. asburiae S-24 showed 190, 183, 176, 154 and 145 nmol h−1 activity,
respectively.

Root growth assay

Microbial activity was evaluated by measuring root growth of MT-tomato and wheat. For
tomato, majority of the bacterial inoculations recorded statistically comparable results to
control. Nevertheless, E. asburiae S-24 significantly increased root length compared to
water-treated controls (Figure 4). For shoot length, highly significant increases of 65%
and 55% were recorded for B. thrugienesis S-50 and M. pluranimalium S-29. For wheat
growth, E. asburiae S-24, B. thrugienesis S-26, M. pluranimalium S-29, B. thrugienesis
S-50 and P. stutzeri S-80 stimulated root length by 45–50% for all strains over un-

RL SL

6 c

5 ab ab ab
a
Length (cm)

4 a
c c
c c
3 bc
a
2

0
S-24 S-26 S-29 S-50 S-80 Control
Strains

Figure 4. Effect of halotolerant rhizobacteria on root and shoot length of MT-tomato. Different
letters on bars indicate significant difference between treatments using Duncan’s multiple range test
(P = 0.05). Abbreviations: RL, root length; SL, shoot length. Affiliations: S-24, E. asburiae; S-26,
B. thuringiensis; S-29, M. pluranimalium; S-50, B. thuringiensis; S-80, P. stutzeri.
1698 A. Raheem and B. Ali

RL SL

18 b b b
b b
16
Length (cm) 14
12 a
10 b
ab bc
8 a ab
6 a
4
2
0
S-24 S-26 S-29 S-50 S-80 Control
Strains

Figure 5. Effect of halotolerant rhizobacteria on root and shoot length of wheat. Different letters on
bars indicate significant difference between treatments using Duncan’s multiple range test (P ˂
0.05). Abbreviations: RL, root length; SL, shoot length. Affiliations: S-24, E. asburiae; S-26, B.
thuringiensis; S-29, M. pluranimalium; S-50, B. thuringiensis; S-80, P. stutzeri.

inoculated control (Figure 5). Similarly, E. asburiae S-24 (33%) and P. stutzeri S-80
(23%) significantly enhanced shoot length.

GUS assay
DR5::GUS activity in MT-tomato was observed after 5 days of seedling exposure to
rhizobacteria. All the bacterial strains recorded positive for GUS activity with slight
variations in the intensity of blue colour that indicated localized auxin accumulation in
leaf and root tissues (Figure 6). Seedlings of MT-tomato showed a greater abundance of
DR5::GUS expression with E. asburiae S-24, B. thuringiensis S-26, P. stutzeri S-80 and
B. thuringiensis S-50 than water-treated control.

a b c

d e f

Figure 6. GUS expression in leaves or roots with halotolerant rhizobacteria in DR5::GUS trans-
genic MT-tomato after 5 days of exposure. (a) E. asburiae S-24, (b) B. thuringiensis S-26, (c)
M. pluranimalium S-29, (d) B. thuringiensis S-50, (e) P. stutzeri S-80 and (f) water-treated control.
 Archives of Agronomy and Soil Science 1699

Bacteria–plant interaction under salt stress

Effect of rhizobacteria on growth of T. aestivum was evaluated in the absence and
presence of different salt stresses (Table 1). In salt-amended soils (without inoculum),
increasing concentrations of NaCl significantly inhibited shoot length and seedling
fresh biomass that is evident from highly significant negative correlation of
r = −0.997; P ˂ 0.05 and r = −0.999; P ˂ 0.05, respectively. However, bacterization
of seeds alleviated salt stress of plants when compared their growth with water-treated
control. For instance, highly significant increases of 40% and 36% were recorded for
shoot length with M. pluranimalium S-29 at 150 mM and 200 mM salt stress.
Similarly, significantly greater shoot elongation was observed for P. stutzeri S-80
(37%), E. asburiae S-24 (35%) and B. thuringiensis S-50 (31%) at 150 mM NaCl.
For seedling fresh biomass, M. pluranimalium S-29 and P. stutzeri S-80 increased
growth by onefold, over control. In case of seedling dry biomass, E. asburiae S-24
and M. pluranimalium S-29 were the most promising to enhance growth by 2.2-fold at
150 mM and 200 mM, respectively. Similarly, P. stutzeri S-80 and E. asburiae S-24
also showed significant increases of 1.9- and 1.2-fold, respectively, for dry biomass at
100 mM and 200 mM.

Table 1. Effect of halotolerant rhizobacteria on the growth of T. aestivum under different salt
stresses.

Fresh Dry weight/

Shoot length weight/five five
Strains NaCl (mM) (cm) seedlings (g) seedlings (g)

Control 0 25.8 (ab) 16.2 (abc) 4.4 (a)

100 25.0 (ab) 15.2 (ab) 4.7 (a)
150 22.4 (a) 14.2 (ab) 4.1 (a)
200 20.4 (a) 12.3 (a) 3.4 (a)
E. asburiae S-24 0 32.8 (bcd) 19.1 (abcd) 10.0 (abcd)
100 32.5 (bcd) 17.0 (abc) 9.7 (abcd)
150 34.9 (d) 25.7 (abcd) 14.0 (cd)
200 32.3 (bcd) 21.9 (abcd) 9.3 (abcd)
B. thuringiensis S-26 0 28.9 (abcd) 18.3 (abc) 5.6 (ab)
100 33.9 (cd) 18.0 (abc) 5.5 (ab)
150 31.5 (bcd) 20.5 (abcd) 6.9 (abcd)
200 33.4 (bcd) 24.2 (abcd) 8.2 (abcd)
M. pluranimalium S-29 0 33.3 (bcd) 24.5 (abcd) 7.1 (abcd)
100 35.1 (d) 24.8 (abcd) 9.1 (abcd)
150 35.2 (d) 15.3 (ab) 6.3 (abc)
200 36.1 (d) 32.8 (d) 14.2 (d)
B. thuringiensis S-50 0 30.5 (bcd) 15.6 (abc) 5.2 (a)
100 34.0 (cd) 13.9 (ab) 5.6 (ab)
150 33.7 (cd) 16.4 (abc) 5.7 (ab)
200 34.4 (d) 18.3 (abc) 6.1 (ab)
P. stutzeri S-80 0 30.5 (bcd) 28.0 (bcd) 6.9 (abcd)
100 21.9 (a) 20.0 (abcd) 8.3 (abcd)
150 35.4 (d) 29.9 (a) 13.1 (bcd)
200 33.9 (cd) 29.8 (cd) 7.3 (bcd)
Note: Mean of 30 plants. Different letters in parenthesis within same column indicate significant difference
between treatments using Duncan’s multiple range test (P = 0.05).
1700 A. Raheem and B. Ali

Table 2. Effect of halotolerant rhizobacteria on vegetative and yield parameters of T. aestivum

under different salt stress.

Shoot Spike
length Number of length Weight of 200
Strains NaCl (mM) (cm) tiller/ plant (cm) seeds (g)

Control 0 72.4 (a) 1.7 (a) 13.8 (bc) 6.9 (bc)

100 68.7 (a) 1.7 (a) 11.1 (a) 6.4 (ab)
150 72.4 (a) 1.7 (a) 13.4 (b) 6.1 (ab)
200 69.5 (a) 1.7 (a) 12.9 (b) 5.5 (a)
E. asburiae S-24 0 82.1 (bcd) 2.4 (bc) 15.1 (def) 9.4 (d–g)
100 78.5 (b) 2.6 (b–f) 14.9 (de) 8.3 (de)
150 82.8 (b–e) 3.0 (c–f) 14.7 (cd) 10.3 (gh)
200 87.1 (d–g) 3.3 (f) 15.2 (def) 9.6 (efg)
B. thuringiensis S-26 0 89.9 (g) 2.9 (b–f) 17.1 (hi) 8.6 (def)
100 88.7 (fg) 3.2 (ef) 16.7 (hi) 8.7 (def)
150 88.8 (fg) 3.0 (e) 16.8 (hi) 9.1 (d–g)
200 85.2 (c–g) 3.1 (def) 15.8 (efg) 9.2 (d–g)
M. pluranimalium S-29 0 81.3 (bc) 2.3 (b–f) 16.7 (ghi) 9.7 (efg)
100 78.4 (b) 2.6 (b–f) 16.0 (fgh) 9.8 (fg)
150 85.6 (c–g) 2.7 (b–f) 17.2 (i) 9.1 (d–g)
200 84.3 (c–f) 2.5 (b–f) 17.6 (i) 9.6 (efg)
B. thuringiensis S-50 0 85.2 (c–g) 2.7 (bcd) 16.9 (i) 9.6 (efg)
100 84.0 (c–f) 2.6 (b–f) 16.9 (i) 8.7 (def)
150 85.8 (c–f) 2.8 (b–f) 16.5 (ghi) 9.4 (d–g)
200 87.8 (efg) 2.7 (b–f) 16.7 (i) 8.7 (def)
P. stutzeri S-80 0 83.1 (b–e) 2.4 (bc) 15.9 (e–h) 11.05 (h)
100 86.1 (c–g) 2.7 (b–f) 17.2 (i) 9.5 (efg)
150 81.3 (bc) 2.7 (b–f) 17.2 (i) 8.1 (cd)
200 81.0 (bc) 2.5 (b–e) 16.5 (i) 9.2 (d–g)
Note: Mean of 30 plants. Different letters in parenthesis within same column indicate significant difference
between treatments using Duncan’s multiple range test (P = 0.05).

At final harvest, majority of the strains have also shown to improve vegetative and
yield parameters of wheat grown under salt stress (Table 2). For instance, B. thur-
ingiensis S-26 and E. asburiae S-24 recorded, respectively, 17–22% and 8–20%
improvements in shoot length in NaCl-amended soil, over water-treated control.
Similarly, number of tillers (up to 94%) and seed weight (up to 40%) witnessed
significant improvements with E. asburiae S-24 and M. pluranimalium S-29 at
200 mM salt stress.

Alleviation of salt stress of T. aestivum by rhizobacteria

Salt-stress alleviation of wheat by rhizobacteria was monitored by analysing sodium
content of plants that were grown at 0 mM, 100 mM, 150 mM and 200 mM NaCl.
After spectroscopic analysis, it was found that sodium content of un-inoculated plant
tissues was increased with increasing salinity levels (Figure 7). On the other hand, salt
stress was significantly alleviated in plants treated with halotolerant rhizobacteria. Overall,
bacterization of seeds reduced salt stress up to 40% (at 100 mM), 58% (at 150 mM) and
61% (at 200 mM) either with B. thuringiensis S-26 or P. stutzeri S-80 when compared
with respective salt-amended control.
 Archives of Agronomy and Soil Science 1701

0 mM 100 mM 150 mM 200 mM

60 e
e
NaCl (mg g–I biomass)

50 d
40 e
c d
c c d b
30 b c b b
a a a a
20 d c
a b a a
10

0
S-24 S-26 S-29 S-50 S-80 Control
Strains

Figure 7. Spectroscopic analysis of salt stress alleviation of T. aestivum by halotolerant rhizobac-

teria in salt-amended soils. Different letters on bars indicate significant difference between respec-
tive salt treatments using Duncan’s multiple range test (P ˂ 0.05). Affiliations: S-24, E. asburiae;
S-26, B. thuringiensis; S-29, M. pluranimalium; S-50, B. thuringiensis; S-80, P. stutzeri.

Discussion
Salinity is one of the major stresses that limit crop growth and productivity. Soil
salinity plays a critical role in the microbial selection process as environmental stress
reduces bacterial diversity. Earlier reports have also indicated that bacteria isolated
from saline areas are more likely to resist inhibitory salt concentrations than their
counterparts from non-saline habitats (Upadhyay et al. 2009). Rhizobacteria that are
residing within the rhizosphere of plants growing in saline habitats may have already
been adapted to salt stress that may be a valuable resource to develop crop inoculants.
In the present study, we isolated rhizobacteria that were producing beneficial plant
growth-promoting metabolites such as IAA and ACC-deaminase activity. In recent
years, several studies have reported the isolation of salt-tolerant rhizobacteria with
agricultural significance (Egamberdieva 2009; Chookietwattana & Maneewan 2012;
Lim et al. 2012). Enterobacter asburiae used in present study is considered potential
human pathogen. Presence of Escherichia coli, Bacillus cereus, Pseudomonas aerugi-
nosa and Staphylococcus saprophyticus has been reported in the rhizosphere with
several plant growth-promoting traits including IAA (Egamberdieva et al. 2008;
Holden et al. 2009; Noreen et al. 2012; Farooq et al. 2014). In addition to beneficial
rhizobacteria, root exudates also attract potential human pathogens that become
enriched in the vicinity of roots where they rapidly utilized simple organic compounds
(Egamberdieva et al. 2008; Holden et al. 2009).
Halophility assays indicated that all the bacterial strains were tolerant to 1M NaCl.
For auxin production, increasing L-tryptophan concentrations recorded significant posi-
tive correlation up to r = 0.991; P ˂ 0.05. Previously, we also demonstrated that
increasing levels of precursor positively stimulated in vitro bacterial auxin production
(Ali et al. 2009b; Akhtar & Ali 2011). On the other hand, amendment of medium with
different salt concentrations showed negative correlation (up to r = −0.992; P ˂ 0.01)
with in vitro auxin production. Nevertheless, lower salinity (0.5M) stimulated auxin
production for E. asburiae S-24, B. thuringiensis S-26 and M. pluranimalium S-29. In
previous studies, we have also demonstrated the auxin production by Halomonas strains
at 1M NaCl (Ali & Hasnain 2007a, 2007b). Similarly, auxin production of several
halotolerant rhizobacteria was unaffected by increasing salinity stresses (Upadhyay et al.
2009; Nakbanpote et al. 2014).
1702 A. Raheem and B. Ali

Biological activity of these microbes was demonstrated by performing rooting and

DR5::GUS assays. Primary root inhibition in MT-tomato or elongation in wheat roots
may also be attributed to auxin synthesis ability of the isolates. Recent studies have
reported the root growth inhibition by rhizobacteria mediated by in vitro production of
increased levels of IAA. The impact of exogenous IAA on root growth ranges from
positive to negative that depends on the amount and sensitivity of plant tissues to IAA
(Hoang et al. 2007; Spaepen et al. 2007; Remans et al. 2008). Auxin response
mediated by rhizobacteria has also been reported by assaying a transgenic auxin-
responsive reporter DR5::GUS in plants (Lόpez-Bucio et al. 2007; Zhang et al. 2007).
High concentrations of salts in soils can reduce plant growth due to large amounts of Na+
having an impact on nutrient availability and inhibition of many enzymes. Salt stress also
inhibits plant growth by disrupting water balance, causing oxidative stress and increased
production of ethylene (Tester & Davenport 2003; Yao et al. 2010; Nakbanpote et al. 2014).
Many strategies have been implicated to improve the salt tolerance of plants, but PGPR-
mediated tolerance against salt stress has been recently focused (Nadeem et al. 2007;
Saravanakumar & Samiyappan 2007; Qin et al. 2014). In the present study, majority of the
bacterial strains exhibiting auxin production and ACC-deaminase activity alleviated salt stress
of T. aestivum in NaCl-amended soils. For instance, M. pluranimalium was the most promising
to enhance shoot length, fresh biomass and dry biomass, respectively, at 150 mM, 150 mM and
200 mM (Table 1). In our experiments, spectroscopic analysis of inoculated plants showed up to
61% reduction in salt stress at 200 mM NaCl. Recently, auxin and ACC-deaminase containing
halotolerant rhizobacteria have been shown to mitigate salt stress with significant impact on
plant growth (Lim et al. 2012; Bharti et al. 2014). Rhizobacteria excrete ACC-deaminase that
degrades the ethylene precursor ACC to ammonia and α-ketobutyrate, thereby reducing the
deleterious levels of stress-induced ethylene. In literature, several studies have indicated that
ACC-deaminase-producing halotolerant bacteria enhanced the growth of plants by mitigating
the salt stress (Mayak et al. 2004; Siddikee et al. 2011; Ahmad et al. 2013). Similarly, bacterial
IAA has also been shown to alleviate salt stress of plants grown under conditions of soil salinity.
For instance, halotolerant bacteria have been reported to enhance the growth by improving the
osmotic stress tolerance in plants (Egamberdieva 2009; Tiwari et al. 2011; Kim et al. 2014).
In conclusion, halotolerant rhizobacteria isolated from Pothohar, salt range of Pakistan
exhibited beneficial plant growth-promoting metabolites. Rhizobacteria have been shown
to produce significant in vitro levels of IAA in NaCl-amended media. Isolates also have
the ability to mediate the auxin response in plants as evident from bioassays with MT-
tomato that suggested their potential use as crop inoculants. Pot trials have shown that
newly isolated strains of E. asburiae S-24, M. pluranimalium S-29 and P. stutzeri S-80
were the most effective in enhancing the growth and yield of wheat in salt-amended soils.
Further, this study also reported the A. arabia growing in saline areas as a potential source
for the isolation of agriculturally important rhizobacteria.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
Higher Education Commission of Pakistan is acknowledged for providing funding to Asif Raheem
under IRSIP No. 1-8/HEC/HRD/2012/2586 to visit Center for Sustainable Agriculture, Lancaster
Environment Center, University of Lancaster, United Kingdom.
 Archives of Agronomy and Soil Science 1703

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