Archives of Agronomy and Soil Science

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Auxin production by rhizobacteria was associated

with improved yield of wheat (Triticum aestivum
L.) under drought stress

Asif Raheem, Alexander Shaposhnikov, Andrey A. Belimov, Ian C. Dodd &

Basharat Ali

To cite this article: Asif Raheem, Alexander Shaposhnikov, Andrey A. Belimov, Ian C. Dodd
& Basharat Ali (2017): Auxin production by rhizobacteria was associated with improved yield of
wheat (Triticum aestivum L.) under drought stress, Archives of Agronomy and Soil Science, DOI:
10.1080/03650340.2017.1362105

To link to this article: https://doi.org/10.1080/03650340.2017.1362105

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Published online: 10 Aug 2017.

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 ARCHIVES OF AGRONOMY AND SOIL SCIENCE, 2017
https://doi.org/10.1080/03650340.2017.1362105

Auxin production by rhizobacteria was associated with improved

yield of wheat (Triticum aestivum L.) under drought stress
Asif Raheema, Alexander Shaposhnikovb, Andrey A. Belimovb, Ian C. Doddc and Basharat Alia
a
Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore, Pakistan; bLaboratory of
Rhizosphere Microflora, All-Russia Research Institute for Agricultural Microbiology, Saint-Petersburg, Russian
Federation; cLancaster Environment Centre, Lancaster University, Lancaster, UK

ABSTRACT ARTICLE HISTORY

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Drought tolerant rhizobacteria of the genus Bacillus, Enterobacter, Received 24 September 2016
Moraxella and Pseudomonas colonizing the root system of Acacia arabica Accepted 27 July 2017
were isolated to mitigate the drought stress of wheat (Triticum aestivum KEYWORDS
L.). In vitro auxin production by rhizobacteria was quantified by Ultra Drought tolerant
High Performance Liquid Chromatography (UPLC). Analysis of the crude rhizobacteria; field capacity;
extracts detected the indole-3-acetic acid (IAA), indole-3-carboxylic acid indole-3-acetic acid; ultra-
(ICA) and indole-3-lactic acid (ILA). Highest IAA production of 25.9 µg performance liquid
ml−1 was observed for Bacillus amyloliquefaciens S-134. Pot trials were chromatography; plant-
conducted to evaluate the role of rhizobacteria to enhance the growth of bacterial interactions; crop
wheat at different water regimes. At highest water stress i.e. 10% field biofertilization
capacity (FC), significant improvement of shoot length was observed
with B. amyloliquefaciens S-134. For yield parameters, B. muralis D-5
and E. aerogenes S-10 recorded 34% and 1 fold increases for spike length
and seed weight, over respective control at 10% FC. Mixed culture
combinations of M-2 (B. thuringiensis S-26, D-2, B. amyloliquefaciens
S-134, B. simplex D-11) and M-3 (M. pluranimalium S-29, B. simplex D-1,
B. muralis D-5, P. stutzeri S-80) showed significant improvement for tillers
and number of spikelets. In conclusion, application of the drought
tolerant rhizobacteria can help to overcome productivity losses in
drought prone areas.

Introduction
Water shortage is one of the major limiting factors that influence crop production. It is estimated
that by 2050, arable land will be seriously affected with drought stress that can cause plant growth
problems (Falkenmark 2013). Drought stress induces various physiological changes in plants which
in turn cause reduction in growth and yield. Crop losses due to water shortage are very common in
arid and semi-arid regions around the world. To cope with water scarcity, various biotechnological
approaches can be employed. Among them is the production of drought tolerant plants through
gene manipulation. Alternatively, plant growth promoting rhizobacteria (PGPR) can be used to
ameliorate drought stress in plants. It is well established from various studies that PGPR help the
plant by triggering their root system to absorb water and nutrients under drought stress (Ngumbi
and Kloepper 2016; Forni et al. 2017).
Several mechanisms have been proposed for rhizobacterial mediated drought stress response in
plants (Kaushal and Wani 2016; Vurukonda et al. 2016). Drought tolerant PGPR positively influence
plant growth by the production of phytohormones, especially auxin (Verma et al. 2010). A majority

CONTACT Basharat Ali basharat.ali.mmg@pu.edu.pk Department of Microbiology and Molecular Genetics, University
of the Punjab, Lahore, Pakistan
© 2017 Informa UK Limited, trading as Taylor & Francis Group
 2 A. RAHEEM ET AL.

of the PGPR has the ability to produce indole-3-acetic acid (IAA) and this is the most abundant type
of auxin produced by rhizobacteria (Ali 2015). For auxin biosynthesis, tryptophan-dependent
pathways are commonly found in microbes. They produce indole-3-acetamide (IAM), indole-3-
acetaldoxime (IAQx), tryptamine (TAM), and indole-3-pyruvic acid (IPA) (Spaepen et al. 2007; Su
et al. 2011). The basic function of auxin is to affect plant cell division, differentiation, initiation of
lateral and adventitious roots. Further, IAA may act as a reciprocal signaling molecule by affecting
gene expression in several bacteria and it also plays a critical role in plant-microbe interaction (Ali
et al. 2010; Cassán et al. 2014; Belimov et al. 2015).
Auxin producing PGPR are better alternative to fight with abiotic stresses. Rhizobacteria have to
modify their phytohormonal activity to induce drought tolerance in plants (Vurukonda et al. 2016).
Plants face various abiotic stresses which badly affect root morphology. It is evident from various studies
that auxin induces the development of adventitious roots to absorb nutrients and water from the
rhizosphere, helping the plant to survive in a stressful environment (Parray et al. 2016). It is also reported
that the synthesis of 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which degrades an ethylene
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precursor, a hormone released during stress, is stimulated by bacterial IAA (Belimov et al. 2009).
In all major crop systems such as wheat, rice, maize and barley, a positive influence from PGPR is
well established (Ali 2015; Bidyarani et al. 2016). Plant growth promotion is mediated by IAA
producing rhizobacteria as it helps plants from seed germination to maturity. It is documented in
the literature that delayed seed germination is due to low levels of phytohormones (Poljakoff-
Mayber et al. 2002). This problem was solved through seed treatment with phytohormone produ-
cing PGPR (Sutariati and Khaeruni 2013). It has been reported that abiotic stresses can lower
phytohormone levels causing retardation of plant growth. PGPR help the plant to compensate for
low levels of hormones by contributing an exogenous source of phytohormones (Ali et al. 2009,
2010). Plant-microbe interactions are also influenced by auxin signaling in common symbiotic
pathways (Laplaze et al. 2015). Moreover, plant hormones have reduced the severity of diseases
caused by bacteria and fungi. For instance, auxin plays a role as a defense molecule in plants
because of its antimicrobial activity (Ludwig-Müller 2015).
For crop production, it is important to develop efficient and cost-effective technologies to manage
abiotic stresses. According to many studies, PGPR help to improve stress tolerance in crop plants
(Belimov et al. 2009; Vílchez et al. 2016). They stimulate physical or chemical changes and activate
mechanisms related to stress tolerance against drought, salinity, nutrient deficiency and plant defense
(Ngumbi and Kloepper 2016). Triticum aestivum L. (wheat) is an important cereal crop. Pakistan is one
of the world’s largest producers of wheat. For the people of Pakistan, wheat is the main staple food
and the country’s most vital commodity. In Pakistan, 80% of farmers grow wheat on 40% total
cultivated area. According to a rough estimate, it contributes about 3.1% value added to GDP and
12.5% to agriculture (Sial et al. 2012). Bacterial inoculations have been shown to stimulate wheat
growth and productivity (Ali et al. 2009; Asghar et al. 2015). Therefore, in the present study, we
isolated drought tolerant rhizobacteria from drought prone areas of Pakistan. We collected the soil
samples from the rhizosphere of Acacia arabica from semi arid localities of the Pothohar plateau of
north-eastern Pakistan. We hypothesized that bacteria colonizing the root system of A. arabica
growing in dry lands may harbor beneficial microbes for agricultural applications. Bacteria residing
within the vicinity of plant roots in such habitats may have naturally adapted to stressful conditions.
Here, we report the potential of Enterobacter, Bacillus, Pseudomonas, and Moraxella strains to enhance
the growth and yield of Triticum aestivum L. under water stress conditions.

Materials and methods

Isolation of drought tolerant rhizobacteria
Drought tolerant rhizobacteria were isolated from the rhizosphere of Acacia arabica that were
growing in different locations of the Pothohar region of Pakistan. In drier or arid woodlands or
 ARCHIVES OF AGRONOMY AND SOIL SCIENCE 3

forests, A. arabica is an important component of vegetation. Bacterial strains that are colonizing the
root system of this plant may have been naturally adapted to tolerate drought stress. Samples were
collected from drought prone rhizospheric soil and brought to the laboratory in sealed polythene
bags. Soil samples were processed within 24 h by making serial dilutions in sterilized distilled
water. About 50 µl of dilution was plated on Tryptic Soya Agar (TSA). TSA agar was supplemented
with four different concentrations of Polyethylene Glycol (PEG6000) i.e., 5%, 10%, 15% and 20%
that imposed −0.03, −0.06, −0.10 and −0.14 MPa osmotic potential, respectively. Pure cultures that
tolerated up to −0.14 MPa were obtained by quadrant streaking and were maintained on L-agar
(Cappuccino and Sherman 2002).

Identification of drought tolerant rhizobacteria

Strains were identified through 16S rRNA gene sequencing. Genomic DNA was extracted from
overnight bacterial cultures using Genomic DNA Purification Kit (Promega, USA). About 1 · 5-kb
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DNA fragment containing 16S rRNA gene was amplified using forward 27F and reverse primers
1522r (Johnson 1994). PCR amplification was performed in 50 µl of Dream TaqTM Green PCR
Master Mix (Fermentas, Waltham, MA, USA) 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
20s at 94°C, primer annealing for 20s at 50°C and extension at 72°C for 2 min. Final extension
was carried out at 72°C for 5 min (Hasnain and Thomas 1996). The amplified products were
purified using QIAquick Gel Extraction Kit (QIAGEN) and sequenced by Eurofins (Lancaster, UK)
using 27F and 1522r primers.

Analysis of bacterial auxin production

A colorimetric method was used for preliminary screening of auxin producing rhizobacteria in the
presence of L-tryptophan. Bacteria were cultivated in L-broth supplemented with 500 µg ml−1 L-
tryptophan for 72 h at 30°C and 150 rpm. An un-inoculated control was kept for comparison. For
each treatment, 3 flasks were inoculated. Bacterial cultures were centrifuged at 2300 × g for 10 min.
Two ml Salkowski’s reagent was added to 1 ml supernatant. Test tubes were incubated in the dark
at room temperature for 30 min as described earlier (Ali et al. 2009). After incubation, optical
density was determined at 535 nm by spectrophotometer (CECIL CE 7200). Bacterial auxin produc-
tion was determined by constructing standard curve against authentic auxin (Sigma-Aldrich, St.
Louis, MO, USA). Supernatants of the same strains were analyzed by Ultra-Performance Liquid
Chromatography (UPLC). The pH of the supernatant was adjusted to 2.5 with 1N HCl. Ten ml of the
cell free supernatant was then extracted thrice with an equal volume of ethyl acetate in 250 ml
separating funnel. Ethyl acetate fraction was separated and evaporated through rotary vacuum
evaporator (Heidolph Instruments, Germany). Dried extracts were dissolved in 18% acetonitrile
(ACN) and stored in 5 ml brown glass vials at −20°C. Extracts were filtered through 0.22 µm nylon
centrifuge tube filters Spin-X (Corning, USA) and fractionated by C18 reverse-phase UPLC (Waters
ACQUITY UPLC BEH Shield RP18 1.7 µm, 2.1–50 mm column) with a 5 µl samples injections. The
UPLC system Waters ACQUITY H-Class (Waters, USA) with fluorescent detector (λex = 280nm,
λem = 350nm) was used for auxin detection. Solvent conditions included a flow rate of 0.3 ml
min−1 in 18% ACN with addition of 0.1% acetic acid. Identification was performed by comparison of
retention times for peaks in the samples and a standard mixture of auxins (Belimov et al. 2015).

Rooting bioassay
Biological activity of bacterial auxin was determined by performing bioassays with wheat (Triticum
aestivum L. var. FD2006). Seeds were obtained from Punjab Seed Corporation (Lahore, Pakistan)
 4 A. RAHEEM ET AL.

and surface sterilized with 0.5% sodium hypochlorite for 5 min. 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 each plate, 10 ml of bacterial culture
adjusted to 107 CFU ml−I were applied and incubated in the dark for 5 days at room temperature.
Water treated seeds were used as control and the experiment was repeated twice with 2 plates per
treatment. After incubation, root and shoot lengths for all treatments were recorded.

Confocal laser scanning microscopy

For confocal scanning laser microscopy (CSLM) wheat seeds were surface sterilized as described
above and soaked in bacterial suspensions (107 CFU ml−I) for 30 min. Then, seeds were placed on
filter paper in disposable petri plates and incubated in an Environmental Test Chamber (MRL-350H;
Sanyo, Osaka, Japan) at 25ºC for 7 days. Seedling roots were excised (5 mm) and stained with
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fluorescent dye (1 mM acradine solution in phosphate buffer, pH 8) for 20 min. Excess stain from
the samples was removed by washing with phosphate and then carbonate buffer (pH 9).
Microscopy was performed using a TCS-NT microscope (Leica, Heidelberg, Germany) equipped
with an Argon-Krypton laser. To visualize the bacterial extracellular colonization, the root surfaces
were observed and a stack of images were generated to a depth of 5–10 μm.

Pot experiments
Pot trials were performed in a natural environment to evaluate the potential of rhizobacteria to
alleviate drought stress of T. aestivum var. FD2006. Pots (30 × 30 cm) were filled with 10 kg loamy
soil. The soil had a pH 7.2; EC 0.45 ds m−I and 0.65% organic matter. Seeds were sterilized as
mentioned earlier and treated with bacterial cell suspension (107 CFU ml−I) for 25 min. For seed
bacterization, drought tolerant bacterial strains that included S-10, S-26, S-29, S-80, S-134, S-137,
D-1, D-2, D-5 and D-11 were used as single cultures (Table 1). For mixed culture inoculations,
combinations of M-1 (S-10, S-80, S-137, D-11), M-2 (S-26, S-134, D-2, D11) and M-3 (S-29, D-1, D-5,
S-80) were used. Initially, 15 seeds were sown in each pot with six replications. After germination,
10 uniform seedlings were maintained. After three and six weeks of germination, plants were
subjected to drought stress by imposing different water stresses i.e., 10%, 15% and 20% lower than
the normal field capacity (FC) of 28% for loamy soil. Moisture level of normal and water deficit pots
was adjusted by adding an appropriate volume of water. Different water stress conditions were
monitored through soil moisture meter SM 150 (Delta, U.K). Pots were arranged in completely
randomized design in the wire house of Department of Microbiology and Molecular Genetics,
University of the Punjab. The average temperature during the growth time period was 20-30ºC
from November to April in Lahore, Pakistan. After 10 weeks of growth period, further thinning was
accomplished to leave 5 seedlings till final harvest. At full maturity, growth parameters such as
shoot length, tillers, spike length, spikelets per spike and grain weight (200 seeds) were recorded.

Table 1. 16S rRNA gene sequencing of bacteria isolated from the rhizosphere of A. Arabica.
S. No. Isolates Identified as Accessions
1 S-10 E. aerogenes S-10 KJ011873
2 S-26 B. thuringiensis S-26 KJ011876
3 S-29 M. pluranimalium S-29 KJ011877
4 S-80 P. stutzeri S-80 KJ011879
5 S-134 B. amyloliquefaciens S-134 KJ011885
6 S-137 B. pumilus S-137 KJ011886
7 D-1 B. simplex D-1 KJ011889
8 D-2 B. thuringiensis D-2 KJ011890
9 D-5 B. muralis D-5 KJ011892
10 D-11 B. simplex D-11 KJ011894
 ARCHIVES OF AGRONOMY AND SOIL SCIENCE 5

Enzyme assays specific to stress tolerance

For proline analysis, fresh leaf material (0.5 g) was extracted with 5 ml of 3% sulphosalicylic acid at
100°C for 10 min with constant shaking. The extracts were filtered through glass wool and analyzed
for proline content using the acid ninhydrin method (Zhang et al. 2009). Racusen and Foote (1965)
method was used for the quantitative estimation of peroxidases. Similarly, for acid phosphatases
the method of Kolari and Sarjala (1995) was used. All the enzyme assays were performed at 10%
and 28% field capacities with 3 replicates for each treatment.

Statistical analysis
Data for in vitro screening, bioassays and pot experiments were subjected to analysis of variance
(ANOVA) using SPSS 20 software and differences between means were evaluated using Duncan’s
multiple range test (P ≤ 0.05).
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Results
Identification of drought tolerant rhizobacteria
The sequences of 16S rRNA gene were analyzed by comparison with sequences in GenBank
through BLAST (http://www.0ncbi.nlm.nih.gov/BLAST). After comparison, strains S-10, S-26, S-29,
S-80, S-134, S-137, D-1, D-2, D-5, and D11 showed around 99% similarity with Enterobacter aero-
genes, Bacillus thuringiensis, Moraxella pluranimalium, Pseudomonas stutzeri, B. amyloliquefaciens, B.
pumilus, B. simplex, B. thuringiensis, B. muralis and B. simplex, respectively. The sequences were
submitted to GenBank and their accession numbers are given in Table 1. Phylogenetic relations
among different bacterial strains are shown in Figure 1.

Auxin production by rhizobacteria

Our colorimetric assay showed that Bacillus strains were the most effective at releasing auxin in
culture medium (Figure 2). For instance, B. pumilus S-137, B. muralis D-5 and B. simplex D-1
produced 167, 151, and 133 µg ml−1 respectively. On the other hand, P. stutzeri S-80 (110 µg
ml−1) and E. aerogenes S-10 (107 µg ml−1) also released significant amount of auxin after 72 h of
incubation. Analysis of crude extracts of rhizobacterial supernatants by UPLC revealed the presence
indole-3-acetic acid (IAA), indole-3-carboxylic acid (ICA) and indole-3-lactic acid (ILA) (Figure 3).
UPLC analysis showed the production of IAA and ILA by all the bacterial strains. Highest levels of
IAA were released by B. amyloliquefaciens S-134 (25.9 µg ml−1), B. muralis D-5 (20.4 µg ml−1) and B.
pumilus S-137 (12.3 µg ml−1). Similarly, maximum ILA levels were recorded with B. simplex D-11
(15.2 µg ml−1), E. aerogenes S-10 (13.4 µg ml−1) and P. stutzeri S-80 (8.43 µg ml−1). Maximum
amount for ICA was observed for B. simplex D-11 (15.2 µg ml−1). However, B. amyloliquefaciens
S-134, B. pumilus S-137 and B. thuringiensis D-2 did not produce ICA (Table 2).

Rooting bioassay
Wheat seed treatment with the strains of Bacillus, Pseudomonas, Moraxella and Enterobacter genera
showed significant improvement for rooting or shooting under in vitro condition (Figure 4). Plant
bacterized with B. simplex D-11, B. thuringiensis D-2 and P. stutzeri S-80 showed 76%, 73% and 66%
increases respectively in root length over controls. Similarly, B. thuringiensis S-26 (74%), E. aerogenes
S-10 (64%) and M. pluranimalium S-29 (61%) were the most effective for shooting.
 6 A. RAHEEM ET AL.

Bacillus thuringiensis S-50 KJ011878

Bacillus thuringiensis D-2 KJ011890
Bacillus thuringiensis S-26 KJ011876
Bacillus marisflavi S-127 KJ011883
Bacillus amyloliquefaciens S-134 KJ011885
Bacillus pumilus S-137 KJ011886
Bacillus pumilus S-151 KJ011888
Bacillus endophyticus S-6 KJ011871
Bacillus endophyticus S-125 KJ011881
Bacillus megaterium S-126 KJ011882
Bacillus megaterium S-145 KJ011887
Brevibacterium frigoritolerans D-3 KJ011891
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Bacillus simplex D-8 KJ011894

Bacillus simplex D-1 KJ011889
Bacillus muralis D-5 KJ011892
Bacillus simplex D-11 KX632093
Enterobacter aerogenes S-10 KJ011873
Enterobacter asburiae S-24 KJ011875
Pseudomonas stutzeri S-80 KJ011879
Prolinoborus fasciculus S-3 KJ011870
Moraxella pluranimalium S-29 KJ011877
Psychrobacter alimentarius S-7 KJ011872
Psychrobacter alimentarius S-11 KJ011874
Psychrobacter alimentarius S-121 KJ011880
Psychrobacter adeliensis S-128 KJ011884

0.05

Figure 1. Phylogenetic relationship among different bacterial strains isolated from drought prone areas. Strains with colored
boxes indicated the drought resistant rhizobacteria used in present study. Strains names are followed by respective accession
numbers submitted to GenBank. The scale bar represents mutations per nucleotide position.

Root colonization
During microscopic analysis, all the parts of root system showed colonization by bacterial strains. Visually,
some strains showed high cellular density on root surfaces as compared to others. For instance, P. stutzeri
S-80 and B. amyloliquefaciens S-134 were recorded to be most efficient in colonizing the root surfaces of
wheat. Figure 5 showed the colonization by a few selected plant growth promoting rhizobacterial strains.

Plant-microbe experiments under drought stress

In the absence of bacterial inoculations, drought stress greatly affected the plant growth and yield.
However, seed bacterization significantly mitigated drought stress and promoted plant growth over un-
inoculated controls. At 10% FC, a significant improvement of shoot length was observed with B.
amyloliquefaciens S-134 (36%) and mixed culture M-3 (28%), over controls (Table 3). On the other hand,
the number of tillers was significantly improved with a mixed culture of M-3 (129%) and E. aerogenes S-10
(76%). For spike length, the mixed culture combination (M-3) and B. muralis D-5 showed a statistically
significant response compared to other treatments. For number of spikelets, 1.5 fold increases were
 ARCHIVES OF AGRONOMY AND SOIL SCIENCE 7

180 d
160 c
140
b b b
Auxin (µg ml-I)

120 b b
100 a
a a
80
60
40
20
0
S-10 S-26 S-29 S-80 S-134 S-137 D-1 D-2 D-5 D-11

Strains

Figure 2. Auxin production by drought tolerant rhizobacteria. Bars represents mean of 3 replicates. Different letters on bars
Downloaded by [University of the Punjab] at 04:44 24 November 2017

indicate significant difference between treatments using Duncan’s multiple range test (≤0.05).

Figure 3. Ultra-Performance Liquid Chromatography (UPLC). Chromatogram showing the production of different indolic
compounds. Red peaks indicate the production of IAA. (a), P. stutzeri S-80; (b), E. aerogenes S-10.
 8 A. RAHEEM ET AL.

Table 2. Production of indolic compounds (µg ml−I) by rhizobacteria after UPLC analysis.
S. No. Strains Indole-3-lactic acid Indole-3-carboxylic acid Indole-3-acetic acid
1 E. aerogenes S-10 0.05 13.4 3.3
2 B. thuringiensis S-26 0.3 0.5 0.4
3 M. pluranimalium S-29 1.2 0.5 0.5
4 P. stutzeri S-80 3.6 8.4 1.9
5 B. amyloliquefaciens S-134 1.0 nd 25.9
6 B. pumilus S-137 14.9 nd 12.3
7 B. simplex D-1 0.4 6.80 2.5
8 B. thuringiensis D-2 14.9 nd 5.6
9 B. muralis D-5 4.6 0.4 20.4
10 B. simplex D-11 2.6 15.2 6.6
Abbreviations: nd, not detected.

Root length Shoot length

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14
d
cd cd cd
12 c
bc c bc
bc bc bc
10 bc b bc ab
ab ab ab ab ab
Length (cm)

8 a
a
6

0
Control S-10 S-26 S-29 S-80 S-134 S-137 D-1 D-2 D-5 D-11
Strains

Figure 4. Effect of bacterial inoculations on rooting and shooting of T. aestivum under in vitro conditions. Mean of 20 plants.
Different letters on bars indicate significant difference between treatments using Duncan’s multiple range test (≤0.05).

Figure 5. Root Colonization of T. aestivum by bacterial strains indicated by confocal laser scanning microscope. (a) E. aerogenes
S-10, (b) P. stutzeri S-80, (c) B. amyloliquefasciense S-134, (d) Control.
 ARCHIVES OF AGRONOMY AND SOIL SCIENCE 9

Table 3. Effect of drought tolerant rhizobacteria on the growth of T. aestivum under natural wire house conditions at 10% field capacity.
Shoot length Tillers/ Spike length Spikelets/ Seed weight/200
S. No. Strains (cm) plant (cm) spike (g)
1 Control 64 (ab) 1.7 (a) 11.8 (a) 24.2 (a) 5.1 (a)
2 E. aerogenes S-10 76 (e) 3.0 (e) 14.6 (bc) 60.2 (cd) 10.2 (h)
3 B. thuringiensis S-26 69 (cd) 2.6 (cde) 14.2 (b) 57.3 (c) 9.3 (f)
4 M. pluranimalium S-29 66 (bc) 2.2 (abc) 13.8 (b) 52.8 (b) 8.2 (e)
5 P. stutzeri S-80 67 (bc) 1.9 (ab) 15.3 (cd) 62.1 (de) 7.7 (d)
6 B. amyloliquefaciens S-134 87 (g) 2.4 (bcd) 15.5 (cd) 52.9 (b) 9.7 (g)
7 B. pumilus S-137 68 (bc) 1.9 (ab) 14.6 (bc) 58.0 (c) 6.9 (c)
8 B. simplex D-1 68 (bc) 1.9 (ab) 14.6 (bc) 58.0 (c) 8.3 (e)
9 B. thuringiensis D-2 76 (e) 2.7 (ab) 14.7 (bc) 51.8 (b) 7.5 (d)
10 B. muralis D-5 66 (bc) 1.8 (ab) 15.8 (d) 67.9 (f) 7.6 (d)
11 B. simplex D-11 72 (d) 2.8 (de) 14.4 (b) 51.3 (b) 6.7 (bc)
12 M-1 61 (a) 2.9 (de) 15.4 (cd) 63.2 (de) 6.9 (c)
13 M-2 68 (bcd) 2.8 (de) 14.6 (bc) 73.9 (g) 6.7 (bc)
14 M-3 82 (f) 3.9 (f) 16.1 (d) 64.9 (ef) 6.5 (b)
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Numeric values indicate the mean of 30 plants. Values followed by different alphabets within the same column indicate
significant difference between different treatments using Duncan’s multiple range test (P ≤ 0.05).
Mixed culture combinations: M-1 (E. aerogenes S-10, P. stutzeri S-80, B pumilus S-137, B. simplex D-11; M-2 (B.
thuringiensis S-26, B. amyloliquefaciens S-134, B. thuringiensis D-2, B. simplex D-11; M-3 (M. pluranimalium S-29, B.
simplex D-1, B. muralis D-5, P. stutzeri S-80.

recorded with M-1, M-3 and P. stutzeri S-80, over respective controls. Similarly, for seed weight, E.
aerogenes S-10, B. amyloliquefaciens S-134 and B. thuringiensis S-26 recorded 100%, 90% and 82%
increases, respectively, over controls. In the case of 15% FC, a significant increase in shoot length over
controls was observed with B. amyloliquefaciens S-134 (20%) and B. simplex D-11 (17%). For yield
parameters, M-1, B. amyloliquefaciens S-134, and M-2 were most effective for tillers (111%), spike length
(19%) and number of spikelets (98%). For seed weight, an 83% increase was recorded with E. aerogenes
S-10 (Table 4). At 20% FC, a majority of the strains, particularly B. amyloliquefaciens S-134 and E. aerogenes
S-10 were significantly improved in shoot elongation at final harvest (Table 5). At normal moisture level
(28% FC), 24%, 190%, 13%, 49% and 71% increases were recorded for shoot length (B. thuringiensis S-26
and B. amyloliquefaciens S-134), tillers (M-2), spike length (B. thuringiensis D-2), number of spikelets (M-3)
and seed weight (E. aerogenes S-10), respectively (Table 6).

Table 4. Effect of drought tolerant rhizobacteria on the growth of T. aestivum under natural wire house conditions at 15%
field capacity.
S. Shoot length Tillers/ Spike length Spikelets/ Seed weight/
No. Strains (cm) plant (cm) spike 200 (g)
1 Control 71 (b) 1.8 (a) 13.7 (a) 32.2 (a) 5.5 (a)
2 E. aerogenes S-10 77 (de) 2.6 (bc) 14.3 (ab) 61.1 (gh) 10.1 (j)
3 B. thuringiensis S-26 79 (ef) 2.8 (c) 14.4 (ab) 59.2 (efg) 10 (j)
4 M. pluranimalium S-29 66 (a) 2.2 (ab) 14.8 (bc) 54.8 (cd) 7.2 (c)
5 P. stutzeri S-80 63 (a) 2.1 (a) 14.6 (bc) 45.2 (b) 8.2 (g)
6 B. amyloliquefaciensS-134 85 (g) 2.8 (c) 16.3 (e) 55.7 (cdef) 9.2 (i)
7 B. pumilus S-137 72 (bc) 2.1 (a) 14.7 (bc) 59.5 (fg) 8.4 (h)
8 B. simplex D-1 74 (bcd) 1.8 (a) 15.5 (cde) 55.4 (cde) 7.9 (f)
9 B. thuringiensis D-2 74 (bcd) 1.8 (a) 14.4 (ab) 55.6 (cde) 6.3 (b)
10 B. muralis D-5 66 (a) 2.1 (a) 15.4 (cde) 47.9 (b) 7.0 (c)
11 B. simplex D-11 83 (fg) 3.1 (c) 15.7 (de) 52.9 (c) 7.6 (de)
12 M-1 62 (a) 3.8 (d) 15.6 (cde) 59.6 (fg) 7.6 (d)
13 M-2 76 (cde) 2.9 (c) 15.1 (bcd) 63.8 (h) 7.8 (ef)
14 M-3 74 (bcd) 3.0 (c) 15.8 (de) 58.4 (defg) 7.7 (de)
Numeric values indicate the mean of 30 plants. Values followed by different alphabets within the same column indicate
significant difference between different treatments using Duncan’s multiple range test (P ≤ 0.05).
Mixed culture combinations: M-1 (E. aerogenes S-10, P. stutzeri S-80, B pumilus S-137, B. simplex D-11; M-2 (B.
thuringiensis S-26, B. amyloliquefaciens S-134, B. thuringiensis D-2, B. simplex D-11; M-3 (M. pluranimalium S-29, B.
simplex D-1, B. muralis D-5, P. stutzeri S-80.
 10 A. RAHEEM ET AL.

Table 5. Effect of drought tolerant rhizobacteria on the growth of T. aestivum under natural wire house conditions at 20% field capacity.
S. Shoot length Tillers/ Spike length Spikelets/ Seed weight/200
No. Strains (cm) plant (cm) spike (g)
1 Control 73 (b) 2.6 (a) 14.1 (a) 43.8 (bc) 6.1 (a)
2 E. aerogenes S-10 92 (fg) 3.2 (a) 15.3 (bcd) 63.1 (fg) 9.5 (i)
3 B. thuringiensis S-26 87 (de) 4.0 (b) 14.8 (ab) 65.2 (fg) 9.5 (i)
4 M. pluranimalium S-29 76 (bc) 2.5 (a) 15.3 (bcd) 44.6 (c) 8.2 (cd)
5 P. stutzeri S-80 83 (d) 3.2 (a) 15.2 (bc) 41.1 (ab) 8.8 (g)
6 B. amyloliquefaciens S-134 94 (g) 4.3 (b) 16.7 (f) 62.3 (f) 9.2 (h)
7 B. pumilus S-137 84 (de) 2.6 (a) 15.5 (bcde) 48.5 (d) 7.8 (b)
8 B. simplex D-1 83 (d) 2.7 (a) 16.4 (ef) 57.5 (e) 10.0 (i)
9 B. thuringiensis D-2 89 (ef) 3.8 (a) 15.6 (bcde) 58.1 (e) 8.4 (ef)
10 B. muralis D-5 74 (b) 3.1 (a) 15.3 (bcd) 38.2 (a) 8.3 (de)
11 B. simplex D-11 88 (ef) 4.2 (b) 16.1 (def) 55.6 (e) 8.0 (c)
12 M-1 83 (d) 4.6 (b) 16.4 (ef) 63.2 (fg) 8.1 (cd)
13 M-2 55 (a) 2.7 (a) 15.3 (bcd) 66.2 (g) 8.1 (cd)
14 M-3 79 (c) 4.5 (b) 15.8 (cde) 62.4 (f) 8.5 (f)
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Numeric values indicate the mean of 30 plants. Values followed by different alphabets within the same column indicate
significant difference between different treatments using Duncan’s multiple range test (P ≤ 0.05).
Mixed culture combinations: M-1 (E. aerogenes S-10, P. stutzeri S-80, B pumilus S-137, B. simplex D-11); M-2 (B. thuringiensis S-26, B.
amyloliquefaciens S-134, B. thuringiensis D-2, B. simplex D-11); M-3 (M. pluranimalium S-29, B. simplex D-1, B. muralis D-5, P.
stutzeri S-80).

Table 6. Effect of drought tolerant rhizobacteria on the growth of T. aestivum under natural wire house conditions at normal
28% field capacity of loamy soil.
S. Shoot length Tillers/ Spike length Spikelets/ Seed weight/200
No. Strains (cm) plant (cm) spike (g)
1 Control 76 (bcd) 2.1 (ab) 15.1 (bc) 46.8 (b) 6.5 (a)
2 E. aerogenes S-10 87 (e) 2.6 (abc) 15.7 (bcde) 61.2 (e) 11.1 (h)
3 B. thuringiensis S-26 94 (f) 2.9 (bc) 14.8 (ab) 66.4 (fg) 10.6 (g)
4 M. pluranimalium S-29 74 (bc) 2.1 (a) 14.2 (a) 46.1 (ab) 8.6 (c)
5 P. stutzeri S-80 81 (d) 2.3 (abc) 15.2 (bc) 42.3 (a) 10.2 (f)
6 B. amyloliquefaciens S-134 94 (f) 3.7 (d) 16.6 (ef) 58.4 (de) 11 (h)
7 B. pumilus S-137 80 (d) 2.9 (c) 15.5 (bcd) 58.9 (de) 7.8 (b)
8 B. simplex D-1 79 (cd) 2.4 (abc) 16.1 (cde) 52.1 (c) 9.8 (e)
9 B. thuringiensis D-2 88 (e) 4.3 (abc) 17.1 (f) 59.1 (de) 9 (d)
10 B. muralis D-5 78 (cd) 2.9 (c) 15.2 (bc) 43.0 (ab) 8.6 (c)
11 B. simplex D-11 89 (e) 4.1 (d) 15.9 (cde) 56.1 (d) 9.6 (e)
12 M-1 76 (bcd) 3.9 (d) 16.4 (def) 65.5 (f) 8.6 (c)
13 M-2 67 (a) 6.1 (e) 15.5 (bcd) 65.1 (f) 9.1 (d)
14 M-3 71 (ab) 4.1 (d) 15.2 (bc) 69.6 (g) 8.7 (c)
Numeric values indicate the mean of 30 plants. Values followed by different alphabets within the same column indicate
significant difference between different treatments using Duncan’s multiple range test (P ≤ 0.05).
Mixed culture combinations: M-1 (E. aerogenes S-10, P. stutzeri S-80, B pumilus S-137, B. simplex D-11); M-2 (B. thuringiensis S-26, B.
amyloliquefaciens S-134, B. thuringiensis D-2, B. simplex D-11); M-3 (M. pluranimalium S-29, B. simplex D-1, B. muralis D-5, P.
stutzeri S-80).

Rhizobacterial influence on enzyme pool of plant under drought stress

Rhizobacterial inoculations showed variable enzymatic contents under different water regimes. At
highest water stress (10% FC), a 6 fold increase in peroxidase activity was observed with mixed
culture M-1, over controls (Figure 6(a)). Bacterization of T. aestivum seed with B. amyloliquefaciens
S-134 and mixed culture M-3 also stimulated peroxidase activity at 28% FC. Maximum acid
phosphatase activity (2-fold) was observed with P. stutzeri S-80 and B. muralis D-5 at 10% FC
(Figure 6(b)). Proline content was increased by B. thuringiensis S-26 (7.4-fold) and mixed culture M-3
(1.6-fold) at 28% and 10% FC, respectively, over controls (Figure 6(c)).
 ARCHIVES OF AGRONOMY AND SOIL SCIENCE 11

a 28% FC 10% FC

4.5 h
4 g h
gh
Peroxidase (mg g-I FW)
3.5 f fg g
3 def fg def ef
de ef d
2.5 cd
c c c
2 c
1.5 b
b
1 ab a
a a a a a
0.5
0
Control S-10 S-26 S-29 S-80 S-134 S-137 D-1 D-2 D-5 D-11 M-1 M-2 M-3

Strains
28% FC 10% FC
b
1.6
d
Acid phosphatase (mg g-I FW)

1.4
d d d
1.2 d d d c
c cd cd
1 c c bc
c bc
ab c c
Downloaded by [University of the Punjab] at 04:44 24 November 2017

0.8 ab ab ab
b b
0.6 a a
a a
0.4
0.2
0
Control S-10 S-26 S-29 S-80 S-134 S-137 D-1 D-2 D-5 D-11 M-1 M-2 M-3

Strains

c 28% FC 10% FC

100 e
90
80
Proline (mg g FW)

70 d e
‐I

60 e
c c c
50 c cd d
40 bc b ab
b b ab ab ab ab b ab b ab b
30 ab a
20 a a
10
0
Control S-10 S-26 S-29 S-80 S-134 S-137 D-1 D -2 D-5 D-11 M -1 M-2 M‐3

Strains

Figure 6. Effect of drought tolerant rhizobacteria on peroxidase, acid phosphatase and proline content of T. aestivum. (a).
Peroxidase, (b). Acid phosphatase, (c). ProlineMean of 3 replicates. Different letters on bars indicate significant difference
between treatments using Duncan’s multiple range test (≤0.05).

Discussion
Drought stress is a recurrent climatic factor faced by plants growing in arid or semi-arid regions. It
can induce nutrient deficiency, lower photosynthesis and hormonal imbalance that lead to low
crop productivity. To manage the food demand, scientists have to go beyond the conventional
agricultural methodologies. One way to do this is rhizosphere engineering (Asghar et al. 2015;
Kaushal and Wani 2016). Previous studies revealed that applications of plant growth promoting
rhizobacteria (PGPR) have the potential to mitigate abiotic stresses (Mayak et al. 2004; Belimov
et al. 2009, 2015; Raheem and Ali 2015; Dessaux et al. 2016). Therefore, in this study, we
investigated the drought stress mitigation of wheat by IAA producing drought tolerant
rhizobacteria.
Screening of rhizobacteria was accomplished on the basis of their auxin production, estimated
by colorimetric as well as UPLC analysis. All the strains showed good potential of IAA production in
L-tryptophan amended medium. UPLC analysis also confirmed the production of three kinds of
auxins that included indole-3-acetic acid (IAA), indole-3-lactic acid (ILA) indole-3-carboxylic acid
(ICA). Production of a variety of indolic compounds by bacteria may indicate the presence of
different pathways for the syntheses of IAA. Belimov et al. (2015) also demonstrated the presence
of indolic compounds in the culture supernatants of P. oryzihabitants Ep4, Variovorax paradoxus 5C-
 12 A. RAHEEM ET AL.

2 and Achromobacter xylosoxidans Cm4. In another study, indole-3-pyruvic acid (IPA), indole-3-
acetaldehyde (IAAld) and indole-3-bytyric acid (IBA) have been reported in bacterial cultures
(Goswami et al. 2015). A variety of indolic compounds in bacterial extracts represent different
pathways used by microbes to produce IAA (Spaepen et al. 2007). In the present study, bacterial
auxin production was taken as major criteria for the selection for effective PGPR for crop
inoculation.
In our rooting assay, strains of Bacillus, Pseudomonas, Moraxella, and Enterobacter showed significant
effects on root growth. This growth response might be related to the production of auxin that has the
ability to trigger root growth. The effect of rhizobacteria on primary and later roots elongation has been
shown to correlate with exogenous levels of microbial IAA (Fibach-Paldi et al. 2012). Similarly, stimulation
of root growth has also been reported by auxin producing rhizobacteria (Aslam and Ali 2015). It was also
demonstrated that increasing auxin concentrations also have the ability to enhance later root growth of
wheat (Ali et al. 2009). In another study, auxin producing rhizobacteria significantly enhanced root
biomass of potato in laboratory assay and tuber yields in field trials under droughty conditions
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(Belimov et al. 2015). The ability of microbes to colonize plant roots is a crucial feature in the study of
plant-microbe interactions. Therefore, we used confocal scanning laser microscopy (CLSM) for a root
colonization study and found a healthy bacterial colonization with the roots of wheat. CLSM enables fast
visualization of bacterial colonization sites on root fragments for small or scattered rhizobacterial
populations (Krzyzanowska et al. 2012).
Drought tolerant rhizobacteria were evaluated in pot trials to mitigate the water stress at different
water regimes. In control plants, a significant reduction in growth parameters was recorded compared to
treated plants. At the highest water stress level, B. amyloliquefaciens S-134 and E. aerogenes S-10 were the
most effective at enhancing vegetative or yield parameters. Hence, we propose that the application of
drought tolerant rhizobacteria is an attractive strategy to ameliorate water stress conditions of plants.
Bacterial phytohormones are one of the possible mechanisms of growth enhancement under harsh
environments (Kaushal and Wani 2016). Timmusk et al. (2014) reported the drought stress amelioration of
wheat by rhizobacteria isolated from drought prone areas. Similarly, inoculation of wheat with drought
tolerant rhizobacteria significantly enhanced vegetative and yield parameters under axenic or filed trials
(Shakir et al. 2012). A negative effect of drought stress has been ameliorated with significant improve-
ments in plant biomass (Kumar et al. 2016). The beneficial rhizobacteria can impart drought tolerance to
plants by the production of exopolysaccharides (EPS), phytohormones, ACC-deaminase activity, volatile
compounds and antioxidants (Vurukonda et al. 2016). In another study, desiccation tolerant bacteria have
been shown to improve the vegetative growth parameter of plants under drought stress (Vílchez et al.
2016). In our study, rhizobacterial inoculations resulted in significant increases in peroxidase, acid
phosphatase or proline content of plants. Exposure of plants to stress conditions led to the production
of reactive oxygen species (ROS) that can cause oxidative damage to macromolecules. Plants and
microbes have the ability to produce enzymatic and non-enzymatic metabolites to detoxify the harmful
effects of ROS (Vurukonda et al. 2016; Forni et al. 2017).

Conclusion
This study revealed that the rhizosphere engineering is a good option to produce environmental
friendly stress tolerance in crop plants. It also indicated the importance of A. arabica growing in dry
lands for the isolation of drought tolerant rhizobacteria for agricultural applications. Rhizobacterial
strains showed significant potential for in vitro auxin production. Isolation of abiotic stress tolerant
rhizobacteria and screening on the basis of PGPR attributes; especially for auxin is a good criterion
for the development of biofertilizers for field trials. Our results showed that the highest positive
effect on vegetative or yield parameters is mainly shown by B. amyloliquefaciens S-134, B. thur-
ingiensis S-26 and E. aerogenes S-10. This study provided new insights to screen drought tolerant
rhizobacteria for biofertilization of agronomic crops growing in dry lands.
 ARCHIVES OF AGRONOMY AND SOIL SCIENCE 13

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 No. 17-5-6
(Bm6-133)/HEC/Sch/2010 and IRSIP No. 1-8/HEC/HRD/2012/2586. This work on the production of auxins by the
bacteria using UPLC was supported by the Russian Science Foundation (14-16-00137).

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