Journal of Plant Physiology 184 (2015) 5767

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Journal of Plant Physiology

journal homepage: www.elsevier.com/locate/jplph

Physiology

The plant-growth-promoting bacterium Klebsiella sp. SBP-8 confers

induced systemic tolerance in wheat (Triticum aestivum) under salt
stress
Rajnish Prakash Singh, Prameela Jha, Prabhat Nath Jha
Department of Biological Sciences, Birla Institute of Technology and Science (BITS), Pilani 333031, Rajasthan, India

a r t i c l e

i n f o

Article history:
Received 21 February 2015
Received in revised form 3 July 2015
Accepted 4 July 2015
Available online 14 July 2015
Keywords:
PGPR
ACC deaminase
Klebsiella sp
Induced systemic tolerance
ERICPCR

a b s t r a c t
Plant-growth-promoting bacteria (PGPB) with 1-aminocyclopropane-1-carboxylatedeaminase (ACCD)
activity can protect plants from the deleterious effects of abioticstressors. An ACCD bacterial strain, SBP8, identied as Klebsiella sp., also having other plant-growth-promoting activities, was isolated from
Sorghum bicolor growing in the desertregion of Rajasthan, India. ACCD activity of SBP-8 was characterized at biochemical, physiological, and molecular levels. The presence of AcdS, a structural gene for ACCD,
was conrmed by the polymerase chain reaction. Strain SBP-8 showed optimum growth and ACCD activity at increased salt (NaCl) concentrations of up to 6%, indicating its potential to survive and associate
with plants growing in saline soil. Inoculation of wheat plants with SBP-8 when grow in the presence of
salt (150200 mM) and temperature (3040 C) stressors resulted inamelioration of stress conditions by
increasing plant biomass and chlorophyll content, and are duction in plant growth inhibition (10100%)
occurred due to salt and temperature stressors. Moreover, strain SBP-8 also caused Na+ exclusion (65%)
and increased uptake of K+ (84.21%) in the host plant. This property can protect plants from adverse
effects of Na+ on plant growth and physiology. Thus, SBP-8 improves growth of the host plant and protects from salt stressors through more than one mechanism including an effect of ACCD activity and on
K+ /Na+ ratio in plants. The colonization efciency of strain SBP-8 was conrmedby CFU (colony-forming
unit) count, microscopy, and ERICPCR based DNA-nger-printing approach. Therefore, and the use of
efcient colonizing plant-growth-promoting bacteria may provideinsights into possible biotechnological
approaches to decrease the impact of salinity and other stressors.
2015 Elsevier GmbH. All rights reserved.

1. Introduction
A continuous increase in the world human population, changes
in lifestyle, urbanization and industrialization have reduced the
total arable land for agriculture. This has thus affected the demand
vs. supply ratio of food. If this issue is not resolved in time, it will
lead to worldwide famine in the future. Moreover, loss of agricultural productivity incurred due to infestation of plant diseases,
damage by insects and nematodes and/or adverse effects of various
abiotic stressors, has been a major concern for agriculture scientists
and economists. Abiotic stressors such as salinity, drought, high
and low temperature, ood, air pollution, heavy metals, organic
contaminants and ultraviolet light reduce the yield of eld-grown

Corresponding author.
E-mail addresses: prabhatn.jha@gmail.com, prabhatjha@pilani.bits-pilani.ac.in
(P.N. Jha).
http://dx.doi.org/10.1016/j.jplph.2015.07.002
0176-1617/ 2015 Elsevier GmbH. All rights reserved.

crops at large scale (Bray et al., 2000). Among these stressors, salinity, uctuation in temperature and drought are the most common
abiotic stressors that severely affect plant growth and production.
Presently, more than 6% of the total land area of the world is saltaffected, especially in arid and semiarid zones (Bui, 2013). The
salinization of land has increased up to 800 million hectares of land
throughout the world, a serious threat to agriculture, suppressing
plant growth and productivity (FAO, 2008; http://www.fao.org/ag/
agl/agll/spush/). Increased salinization of land in this manner will
result in up to 50% land loss by the middle of the twenty-rst century (Wang et al., 2003). Similarly, it is estimated that the total land
area affected by drought will increase two-fold and water resources
will decline by 30% by the year 2050 (Falkenmark, 2013). Moreover,
there is evidence of yield declines in wheat and paddy crops in many
parts of South Asia due to increasing water stress and increased air
temperature. The average temperature on the Indian sub-continent
has risen by 0.57 C in the last 100 years and is likely to increase
a maximum of 2.5 C by 2050 and 5.8 C by 2100 (Venkateswarlu

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R.P. Singh et al. / Journal of Plant Physiology 184 (2015) 5767

et al., 2008). Problems occurring due to these stressors can be overcome to a certain extent by developing stress tolerant transgenic
plants. However, issues such as high cost, loss of genes and other
regulatory issues associated with transgenic plants are important
bottlenecks for releasing these plants at the eld level (Glick, 2007).
Therefore, the application of certain plant growth-promoting rhizobacteria (PGPR) to recover plant growth under such stressful
conditions has been proposed in recent years to be more effective
and sustainable.
PGPR can improve plant growth through one or more mechanisms, either directly through providing nitrogen, phosphate, iron
nutrition and production of phytohormones, or indirectly through
protection from pathogenic and insect pests by antagonistic mechanisms or generating induced systemic resistance in host plants. In
addition, certain PGPR can also protect plants from abiotic stressors through induced systemic tolerance (IST), which enables plants
to tolerate or attenuate deleterious effects of abiotic stressors
(Yang et al., 2009). One of the most common mechanisms of IST
is achieved through the production of ACC (1-aminocyclopropane1-carboxylate) deaminase by the plant-associated bacteria. Under
abiotic or biotic stress conditions, plants synthesize ethylene,
which at higher concentrations (termed as stress ethylene) inhibits
elongation of the plant shoot and root, causes chlorosis and leaf
abscission, suppress leaf expansion or promotes epinasty (Abeles
et al., 1992). ACC deaminase produced by associated bacteria
reduces the level of stress ethylene by degrading ACC, an immediate precursor of ethylene, to ammonia and -ketobutyrate and
promotes plant growth under stress conditions. Breakdown products of ACC serve as the source of nitrogen and energy for associated
bacteria (Glick, 2007). Experimental evidence suggests that these
bacteria promote plant growth in terms of osmotic balance, altering
root size and morphology and increase nutrients uptake adjusting
the nitrogen metabolism (Compant et al., 2005). Different bacterial
genera possess different levels of enzyme activity under various
environmental conditions. However, organisms with ACC deaminase activity of approximately >20 nmol -ketobutyrate mg1 h1
are sufcient to promote plant growth (Penrose and Glick, 2003).
Recently, use of ACC deaminase possessing bacteria has become
a promising alternative for plant growth promotion and alleviation of plant stress caused by salinity (Jha et al., 2012; Karthikeyan
et al., 2012; Rashid et al., 2012). For instance, ACCD bacteria Achromobacter piechaudii ARV8 was found to be efcient in promoting the
growth of tomato under salt stress (Mayak et al., 2004). An increase
in seedling growth, biomass and reduction of stress ethylene (57%)
in red pepper was observed after inoculation of ACC deaminase
bacteria (Siddikee et al., 2011). Similarly, Pseudomonas uorescens
strain TDK1 with ACCD activity enhanced salt resistance in groundnut plants as compared to other Pseudomonas strains having no ACC
deaminase activity (Saravanakumar and Samiyappan, 2007). Thus,
salt tolerant ACCD containing bacteria could be advantageous in a
saline environment to deliver benecial effects on plants
In addition to production of stress ethylene, several biochemical
processes such as protein synthesis and photosynthetic pigments
are severely affected by increased salinity (Parida and Das, 2005).
Higher accumulation of salt (Na+ ) ions reduces the K+ uptake by
roots of higher plants (Maathuis et al., 2014). A number of experimental studies have demonstrated that salinity negatively affects
the Ca2+ absorption and its translocation in plants (Liu et al., 2014;
Zhang and Shi, 2013). Plant-growth-promoting rhizobacteria ameliorate the ion-induced damage and improve plant growth through
HKT1 (high afnity K+ transporter) gene expression in saline conditions. The differential regulation of HKT1 gene expression reduces
the aggregation of Na+ and increases the accumulation of K+ in
shoot and roots of Bacillus subtilis GB-03 inoculated Arabidopsis
seedlings under salt stress (Zhang et al., 2008). Hence, these PGPR
act as important promoters for HKT1 gene expression (Alizadeh and

Namazi, 2011). The HKT transporter has been found in many plant
species. However, the regulatory mechanism is still not known
(Horie et al., 2008). Understanding the mechanism of development
of salt tolerant plants either by genetic engineering or use of plantgrowth-promoting bacteria is essential for solving the problem of
productivity in salty regions (Dimkpa et al., 2009).
The isolation of ACCD bacteria and their benecial effect on plant
growth under various environmental stressors have been reported
by several research groups (Ahmad et al., 2011; Cheng et al., 2007;
Shaharoona et al., 2007). Soil salinity is one of the most important factors adversely affecting soil microbial activities and crop
productivity. Enhancement of growth and salt tolerance in PGPR
inoculated red pepper (Siddikee et al., 2011), tomato (Mayak et al.,
2004) and groundnut (Saravanakumar and Samiyappan, 2007) have
been reported. However, little information is available on the effectiveness of ACCD bacteria on growth of wheat plant, one of the most
economically important crops, under salt stress (Nadeem et al.,
2006). Wheat (Triticum aestivum), belonging to the family Graminaceae, is an annual, large shrub plant commonly used as staple
food. Wheat growing under eld conditions is exposed to various
biotic and abiotic stressors, including high salt, high temperature and drought at various developmental stages. Therefore, the
present work aimed to isolate efcient ACCD bacteria from plants
growing in saline soil of desert of Rajasthan, India and to characterize them for their plant growth stimulating effect on wheat plant
under salt and temperature stress conditions.
2. Materials and methods
2.1. Isolation of ACC deaminase bacteria
ACCD bacteria was isolated from the rhizospheric soil of
Sorghum bicolor growing in the desert region of Rajasthan, India
(28 18 N, and 74 58 E). For the isolation, one gram of soil adhered to
roots was added to 50 ml sterile PAF media (Composition: per litre,
10 g proteose peptone, 10 g casein hydrolysate, 1.5 g anhydrous
MgSO4 , 1.5 g K2 HPO4 and 10 ml glycerol) and incubated in a shaker
at 200 rpm at 30 C for 24 h. 1 ml culture was then transferred to
50 ml sterile DF (Dworkin and Foster) minimal salt medium supplemented with 3.0 mM ACC (SigmaAldrich Co. Mumbai, India).
Composition of DF medium was as follows (per litre): KH2 PO4
4.0 g, Na2 HPO4 6.0 g, MgSO4 7H2 O 0.2 g, glucose 2.0 g, gluconic acid
2.0 g, citric acid 2.0 g, Trace elements: FeSO4 7H2 O 1 mg, H3 BO3
10 g, MnSO4 H2 O 11.19 g, ZnSO4 7H2 O124.6 g, CuSO4 5H2 O
78.22 g, MoO3 10 g, pH 7.2 (Dworkin and Foster, 1958). After two
rounds of enrichment in above media, dilution of this culture was
plated on solid DF salt minimal medium spread with 3 mM ACC
(SigmaAldrich, USA) and incubated for 48 to 72 h at 30 C. Bacteria growing on above selective medium were sub-cultured several
times on DF-ACC plate for conrming ability of isolates to utilize
ACC as nitrogen source. Based on higher ACCD and other plantgrowth-promoting activities, SBP-8 was selected for detailed study.
Selected ACCD positive isolates were preserved for screening of ACC
deaminase assay and other plant growth stimulation properties.
Glycerol stock (15% w/v) of the isolate was prepared and stored at
70 C until further use.
2.2. ACC deaminase assay
2.2.1. Preparation of enzyme extract
ACCD activity was assayed according to the method of Honma
and Shimomura (1978) with slight modications. Bacterial isolate
SBP-8 was cultured in tryptic soya broth (Himedia Laboratories,
Mumbai, India) and grown up to late log phase in a shaking water
bath at 200 rpm at 30 C. The accumulated biomass was harvested

R.P. Singh et al. / Journal of Plant Physiology 184 (2015) 5767

by centrifugation at 8000 g for 10 min at 4 C. Bacterial pellets were

washed with DF salt minimal medium and suspended in DF salt
minimal medium with ACC of nal concentration 3.0 mM. The bacterial cells were returned to shaking water bath for induction of
ACCD at 200 rpm for 24 h at 30 C. Then cells were harvested by
centrifugation, washed with 0.1 M TrisHCl (pH 7.6). The collected
bacterial cells were resuspended in 0.1 M TrisHCl (pH 8.5). Thirty
microlitre of toluene was added to cell suspension and vortexed
at highest setting for 30 s. At this point 100 l of toluenized cells
were set aside for protein assay at 4 C. The remaining toluenized
cell suspension was used immediately for ACCD assay.
2.2.2. ACCD assay
20 l 0.5 M ACC was added to 200 l toluenized cells, briey
vortexed and incubated at 30 C for 15 min. Following this, 1 ml
of 0.56 M HCl was added, mixed by vortexing and spun for 5 min
at 8000 g at room temperature. One ml of resulting supernatant
was mixed with 800 l of 0.56 M HCl. Then 300 l of 2, 4dinitrophenylhydrazine was added to the above mixture in a glass
tube and incubated at 30 C for 30 min. Following the addition and
mixing of 2 ml of 2N NaOH, the absorbance was measured at 540 nm
in a spectrophotometer (Jasco Corporation, Japan). The number of
mol of -ketobutyrate (-KB) produced was determined by comparing the absorbance at 540 nm of a sample to a standard curve of
-ketobutyrate ranging between 0.1 and 1.0 mol.
2.3. Screening for plant-growth-promoting properties
2.3.1. Phosphate solubilization assay
A phosphate solubilization assay of the isolate was performed
following the method of Mehta and Nautiyal (2001) on National
Botanical Research Institutes Phosphate (NBRIP) medium containing insoluble tricalcium phosphate. Freshly grown culture of the
bacterial isolate was point inoculated on media and kept at 28 C
for 4 days. The clear zone around the inoculated culture was considered as positive for phosphate solubilization activity. Solubilized
Phosphate was quantied according to the method of Ames (1964),
keeping K2 HPO4 as standard.
2.3.2. Production of phytohormones
Selected isolate was tested for production of phytohormones
indole-3-acetic acid and gibberellic acid. Production of IAA was
tested and quantied using the method of Gordon and Weber
(1951). Briey, three days old culture grown in NB (Nutrient
broth) containing 100 g ml1 tryptophan at 30 C with shaking
(180 rpm), was harvested by centrifugation at 9000 g for 2 min.
In tubes, 1 ml supernatant of the culture was mixed with 4 ml of
IAA reagent (Salkowskys) and kept for 30 min at room temperature. Development of pink color shows a positive test for IAA.
Optical density was measured spectrophotometrically at 530 nm
using a Jasco-630 UVvisible spectrophotometer (Jasco Corporation, Japan). The concentration of IAA in each sample was
determined from the standard curve of IAA. Uninoculated media
was used as a control.
The estimation of gibberellic acid was performed by the spectrophotometric method following the protocol of Holbrook et al.
(1961). The bacterial isolate was grown in 100 ml NB medium at
28 C for 3 days. Thereafter, the culture was centrifuged at 8000 g
for 10 min. The pH of the supernatant was adjusted to 2.5 using
1 N HCl and it was extracted with equal volume of ethyl acetate in
a separating funnel. The extract was transferred to another separating funnel and retreated with the equal volume of ethyl acetate
23 times to get a maximum amount of gibberellic acid. To 1.5 ml
of extract, 0.2 ml of potassium ferrocyanide was added and centrifuged at 1500 g for 10 min. An equal volume of 30% HCl was added
in the supernatant and incubated for 1 h at room temperature. The

59

absorbance of the mixture was measured at 254 nm in a UVvisible

spectrophotometer. The amount of gibberellic acid was calculated
from the standard curve (10100 g ml1 ).
2.3.3 Assay for ammonia production
Freshly grown culture of bacterial isolate was inoculated into
10 ml peptone water in separate tubes and incubated for 48 h at
37 C. After the bacterial growth, Nesslers reagent (0.5 ml) was
added to each tube. Development of brown to yellow color was
observed as a positive test for ammonia production (Cappuccino
and Sherman, 1992). Uninoculated medium was used as blank for
comparison.
2.3.4. Siderophore production
Siderophore production of the isolate was tested by chrome azurole S agar (CAS) method described by Schwyn and Neilands (1987).
Spot inoculation of the test organism was done on the chrome azurole S agar plate and incubated at 30 C for 45 days. Development
of yelloworange halo zone around the colony was considered positive for siderophore production.
2.3.5. Test of antagonistic activity
Antagonistic activity of the isolate SBP-8 was determined
by using agar well diffusion method against important plant
pathogenic fungal species namely Aspergillus avus, Fusarium
oxysporum, Fusarium moniliforme and Fusarium graminearum.
Antagonistic activity against certain bacterial pathogens such as
Bacillus cereus, Erwinia carotovora, Escherichia coli, and Staphylococcus aureus was also determined. Briey, freshly grown cultures of
selected fungal and bacterial species were spread on tryptic soya
broth and potato dextrose agar plates, respectively. After adsorption, well size of 6 mm was made by metallic borer and lled with
108 CFU/ml of a freshly grown culture of SBP-8. The plates were
incubated for 7 days at 28 C for fungal species and 24 h at 37 C
for bacteria. Boiled culture of microbial species was used as a control. Antagonistic activity was determined by measuring the zone
of inhibition for which parameter used was <10 mm = poor (+),
between 10 and 20 mm = good (++).
2.3.6. Screening for salt and temperature tolerance
The isolate was screened for their ability to tolerate salt stress.
For that purpose, 20 l of overnight grown culture was inoculated
into DF media supplemented with different concentrations of NaCl
(0.510%). For the test of temperature tolerance for a given isolate, it
was grown in NB medium at different temperatures (2550 C). Cultures were grown for 24 h and growth of cultures was determined
by measuring absorbance at 600 nm using uninoculated broth as a
blank. Each experiment was performed in triplicate.
2.3.7. Biochemical characterization
Biochemical tests including Gram staining, a starch agar test,
an IMViC (Indole, Methyl Red, Voges Proskauer, Citrate utilization) test and a catalase assay were performed following standard
protocol (Harley and Prescott, 2002). The ability of the isolate
to utilize different carbohydrates was tested using a carbohydrate utilization test kit (KB 009, Himedia) as per instruction of
the manual. It was also tested for its resistance against standard antibiotics, namely gentamicin (30 g), ampicillin (10 g),
erythromycin (10 g), kanamycin (5 g), tetracycline (10 g),
streptomycin (25 g), and chloramphenicol (10 g) by the antibiotic sensitivity assay using antibiotic discs (HTM 002, Himedia).
The experiment was performed in triplicate. The results were interpreted on the basis of the diameter of inhibition zone using the zone
size interpretative chart supplied by the manufacturer (Himedia,
India).

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R.P. Singh et al. / Journal of Plant Physiology 184 (2015) 5767

2.3.8. PCR amplication of bacterial 16S rRNA gene and

sequencing
Total genomic DNA of the bacterial isolate was extracted
using genomic DNA extraction kit (Qiagen, USA). Amplication of 16S rRNA gene was done using universal primer
27F1 (5 -AGAGTTTGATCMTGGCTCAG-3 ) and 1494Rc (5 TACGGCTACCTTGTTACGAC-3 ) where M designates A or C bases.
PCR reaction was carried out in a 25 l reaction mixture containing
10 X buffer (with 15 mM MgCl2 ) 2.5 l, 10 pmole forward and
reverse primers, dNTP mixture (2.5 mM each) 3.0 l, 0.5 l of Taq
DNA polymerase (3 U L1 ) and 50 ng of DNA template. Volume
was maintained by nuclease free water (Genetix, India). DNA samples were amplied on DNA thermal cycler (T100, Bio-Rad, USA).
The PCR conditions were as follows: initial denaturation for 3 min
at 94 C, 30 cycles each consisting of denaturation for 1 min at 94 C,
primer annealing for 1 min at 54 C and extension at 72 C for 5 min
and a nal elongation of 5 min at 72 C. PCR amplied products
were analyzed by electrophoresis on 1% agarose gel stained with
0.5 g ml1 ethidium bromide. Sequencing of puried PCR product
was done by Xcelris Genomics Labs Ltd. (Xcelris, Ahmedabad)
India. The sequenced nucleotides were compared against GenBank
database using the NCBI BLAST algorithm and deposited in the
NCBI database http://www.ncbi.nlm.nih.gov/BLAST Taxonomic
afliation of SBP-8 was assigned using RDP database (http://rdp.
cme.msu.edu/seqmatch/seqmatch intro.jsp) at 98% threshold of
16S rRNA gene sequence. A phylogenetic analysis was done by
using software MEGA6.0 (Tamura et al., 2013) and aligned using
CLUSTAL-X. The pairwise evolutionary distance was constructed
by neighbor-joining method with the bootstrap of 500 replicates
to cluster the associated taxa.
2.3.9. Physiological characterization of ACCD activity
The effects of various physiological conditions such as temperature and salt concentrations on ACCD activity of Klebsiella sp. SBP-8
were also tested in vitro. Bacterial culture was grown in DF minimal
media supplemented with ACC and ACCD activity was measured
as described above. Media or culture condition was modied for
different treatments as follows. Bacterial culture was grown at
different temperatures ranging from 25 C to 40 C for evaluating
the effect of temperature on ACCD activity. Similarly, 28% NaCl
was used to assay ACCD activity under different salt treatment.
For assessment of ACCD activity in various carbon sources, 0.2%
each of glucose, sucrose, lactose, xylose, and dextrose was added
in DF medium. Ammonium nitrate, ammonium chloride, ammonium sulfate and potassium nitrate (0.2%) were added separately
in DF medium as nitrogen sources. Concentration of substrate (ACC)
ranged between 1 and 5 mM. The pH of the medium was adjusted
between pH 5.0 and pH 9.0 to test the effect of different pH on
enzyme activity. Similarly, enzyme activity was assessed at different time intervals also. Cultures of equal OD (OD600) were used for
ACCD assays.
2.3.10. Amplication, sequencing and analysis of AcdS gene
AcdS gene encoding ACCD was amplied by PCR using
a universal pair of primers designed by Duan et al.
(2009). Sequences of primers were as follows: Forward
(F) 5 -GGCAAGGTCGACATCTATGC-3 and Reverse (R) 5 GGCTTGCCATTCAGCTATG-3 . Amplication was performed in
a nal volume of 50 l reaction mix containing 50 ng genomic DNA
(template), 20 pmole each of forward and reverse primers, 200 M
each dNTPs (Genei), 1 X Taq polymerase buffer with 1.5 mM MgCl2
and 2.5 U Taq DNA polymerase (Genei). The PCR was carried out
in a thermal cycler (T100, BioRad, USA). The reaction conditions
of PCR included an initial denaturation step at 94 C for 3 min, 30
amplication cycles of denaturation at 94 C for 1 min, annealing
at 58 C for 1 min and primer extension at 72 C for 3 min, followed

by a nal extension at 72 C for 5 min. The PCR puried amplicon

of AcdS gene was sequenced (Xcelris Genomics, Ahmedabad,
India). The identity of AcdS amplicon was conrmed using the
BLAST algorithm available at http://www.ncbi.nlm.nih.gov/BLAST
phylogenetic analysis was done as described for rRNA sequence
analysis.
2.3.11. Test of motility
Because motility is required for colonization, motility of SBP-8
was tested using standard protocol (Connelly et al., 2004).
(i) Swimming: Tryptone swim plates (1% tryptone, 0.5% NaCl, 0.3%
agar) were inoculated with a sterile toothpick and incubated
for 16 h at 25 C. Motility was then assessed qualitatively by
examining the circular turbid zone formed by the bacterial cells
migrating away from the point of inoculation.
(ii) Swarming: Swarm plates were composed of 0.5% Bacto Agar
and 8 g of nutrient broth/liter (both from Difco, USA) supplemented with 5 g l1 of dextrose and dried overnight at room
temperature. The cells were point inoculated with a sterile
toothpick and the plates were incubated at 30 C for 24 h.
(iii) Twitching: The cells were stab inoculated with a toothpick
through a thin (approximately 3 mm) LB agar layer (1% agar)
to the bottom of the Petri dish. After incubation for 2448 h at
30 C, a hazy zone of growth at the interface between the agar
and the polystyrene surface was observed.
2.4. Effect of strain SBP-8 on plant growth under salt and
temperature stress
2.4.1. Physiochemical characteristics of soil
The soil sample used for the pot study was analyzed for its various physiochemical properties. pH and electrical conductivity of
soil were analyzed by digital pH and EC meter on an 1:2.5 ratio
of soil and water suspension. The estimation of organic carbon
was done using the method of Walkley and Black (1934) using
1N potassium dichromate for titration and 0.5N ferrous ammonium sulfate for back titration. Available soil phosphorus (Olsen
P) was determined by chlorostannus-reduced molybdophosphoric
blue color method after extraction with 0.5 M sodium bicarbonate
as described by Olsen et al. (1954). Available nitrogen and potassium and other micronutrients (Fe, Cu, Zn, and Mn) were estimated
by the method of Jackson (1967).
2.4.2. Preparation of bacterial inoculum and seed treatment
On the basis of growth promoting properties shown by isolate SBP-8, its effect on plant growth under salt and temperature
stressors was tested on the wheat plant (Triticum aestivum C-309)
in a controlled environment. Preparation of bacterial inoculum
and seed treatment were performed according to Penrose and
Glick (2003) with slight modications. Briey, freshly grown culture (30 ml) was harvested and suspended in 0.5 ml sterile 0.03 M
MgSO4 and diluted in the same solution to adjust absorbance to
0.15 at 600 nm. Wheat seeds were surface sterilized by treating
with 70% ethanol for 2 min followed by three times washing with
sterilized water. Then, the seeds were exposed to 1.0% sodium
hypochlorite (NaOCl) solution for 3 min followed by three consecutive washes using sterile water to remove all traces of sodium
hypochlorite. Each Petri dish containing around fteen seeds was
incubated at room temperature with appropriate treatment: sterile 0.03 M MgSO4 control and bacterial suspension in sterile 0.03 M
MgSO4 (OD of 0.15 at 600 nm). Bacterized seeds were air dried for
one hour under aseptic conditions. Following incubation period,
ve seeds were sown in plastic pots lled with sterile soil in triplicates in a growth chamber with 16:8 photo-period at 28 2 C.
Hoagland media was applied to soil as a nutrient solution on alter-

R.P. Singh et al. / Journal of Plant Physiology 184 (2015) 5767

nate days (Hoagland and Boyer, 1936). For imposing salt stress,
plants were watered with 0 mM, 150 mM, 175 mM, and 200 mM
salt solution at 48 h intervals. Pots were arranged in a completely
randomized block design with three replicates in each treatment.
For temperature stress, bacterized seeds were grown at different
temperatures (25 C, 30 C, 35 C, 40 C) maintained in the growth
chamber. Plants were harvested 15 days after the sprouting of the
seeds and plant growth was measured based on various parameters
such as percentage of germination, root and shoot length, fresh and
dry weight of ve randomly selected seedlings and chlorophyll content. For measurement of Chlorophyll a/b, fresh leaf samples (1 g)
were homogenized in 80% acetone and pigments were extracted
and quantied (Duxbury and Yentsch, 1956). The absorbance at
480, 510, and 663 nm was measured on a UVvis spectrophotometer (Jasco, Japan). Plants from the same experimental setup were
also used for estimation of Na+ , K+ , Ca2+ and colonization studies
as described below.
2.4.3. Estimation of Na+ , K+ , Ca2+ in plant tissues
Shoots were washed with autoclaved Milli-Q water ve to six
times and oven dried at 70 C for 48 h. Subsequently 1 g of shoot tissue was ground in liquid N2 and digested in a mixture of 30% H2 O2 ,
65% HNO3 and deionized water in a ratio of 1:1:1 at 120 C for 2 h
to a nal volume of 12 ml in a microwave digester. The nal volume was adjusted to 20 ml with deionized water. Ions Na+ , K+ and
Ca2+ were estimated by an atomic absorption spectrophotometer
(AAS 2380, PerkinElmer, USA) at National Horticultural Research
and Development Foundation (Nashik, India). For accuracy, each
sample was analyzed in triplicate sets.

2.4.4. Test of colonization efciency and ERIC PCR

The plants were surface-sterilized and cut into small sections of
0.3 0.4 cm. The sections were vigorously vortexed with the glass
beads in sterilized PBS buffer and plated onto nutrient agar media
and incubated for 24 days at 30 C. The colonies were counted
using plate dilution method and represented as cfu g1 fresh tissue. For colonization study, 12 cm of root segments was stained
in a solution of 0.1% acridine orange for 23 min followed by washing thrice with sterile distilled water. The stained root was placed
on a glass slide with a cover slip on top of it and viewed under
epiuorescence microscope (Olympus-CKX41, Olympus, Japan) at
intensity between 450 and 490 nm using 100X objective lens and
10 X eyepiece lens.
For conrming identity of colonized bacteria, ERICPCR was
performed to amplify DNA fragments containing enterobacterial
repetitive intergenic consensus (ERIC) sequence. On the 15th day
of plant growth, total DNA was extracted from plant root by
plant genomic DNA extraction kit (Himedia, India). Primers of
ERIC 1R F (5 -ATGTAAGCTCCTGGGGATTCAC-3 ) and ERIC 2R (5 AAGTAAGTGACTGGGGTGAGCG-3 ) were used for amplication.
The PCR reaction was performed in a 50 l reaction volume containing 50 ng (3 l) of template, 125 M each dNTPs (Bangalore
Genei, India), 1.5 mM MgCl2 , 20 pmol of each primer and 1.5U of
Taq DNA polymerase in a DNA thermal cycler (T100, Bio-Rad, USA).
The cycling condition was: initial denaturation for 5 min at 94 C,
30 cycles each consisting of denaturation for 1 min at 94 C, primer
annealing for 1 min at 52 C and extension at 72 C for 5 min and a
nal extension of 5 min at 72 C. Analysis of amplied products was
done on 1.8% agarose gel containing 0.5 g ml1 ethidium bromide
using gel documentation system (Bio-Rad, USA).

2.4.5. Statistical analysis

All of the experiments were conducted in triplicate and results
were tabulated as mean standard deviation (SD). Data were

61

analyzed by analysis of variance (ANOVA) and subsequently by

Duncans multiple range test at p = 0.05.
3. Results
3.1. Isolation and primary characterization of ACC deaminase
bacteria
Actively growing bacterial colonies on DF medium supplemented with ACC as a nitrogen source were selected and
sub-cultured several times. ACCD activity of selected isolate SBP8 was further conrmed by ACCD assay. ACCD activity of the
SBP-8 was measured and found to be 396.20 21.18 nmol of ketobutyrate mg1 Pr. (protein) h1 .
The isolate SBP-8 was further subjected to certain biochemical
tests and 16S rRNA gene-based sequence analysis for identication.
It was found positive for the test of catalase, VP (Voges-Proskauer),
lipase and nitrate reductase whereas negative for indole, MR
(methyl red), amylase and urease. The tolerance of this isolate to
salt and temperature stress was also assessed. It showed growth up
to 50 C of temperature and the optimal temperature for the growth
of SBP-8 was 30 C. SBP-8 showed optimal growth up to 6% of NaCl,
while it could tolerate up to 8% NaCl concentration in the growth
medium. The isolate was able to utilize various carbon sources
(Suppl. Table 1). Moreover, antibiotic sensitivity proling of SBP8 showed resistance to kanamycin, vancomycin, tetracycline and
gentamicin but sensitivity to chloramphenicol. For identication
of isolate at the molecular level, 1.5 kb amplicon of 16S rRNA gene
was obtained by PCR. The sequence of resulting amplicon was submitted to the NCBI Genbank under the accession number KJ950709.
Based on the BLAST result, the strain was identied as Klebsiella sp.
having a closest match of 96% similarity to Klebsiella pneumoniae
HKG219. Dendrogram results also showed its similarity to other
strains of K. pneumoniae (Fig. 1).
3.2. Molecular characterization of AcdS gene
It is evident from Fig. 2A that a 650 bp amplicon specic to
AcdS, a functional gene encoding ACCD, was obtained from genomic
DNA of Klebsiella sp. SBP-8 by using a specic pair of primers. On
performing gene alignment analysis using BLAST algorithm, identication of given amplied fragment as AcdS was conrmed with
99% sequence similarity with AcdS gene of K. pneumoniae AcdSPB 2.
The obtained sequence of isolated SBP-8 was submitted to the NCBI
GenBank database under the accession number KM501058. Phylogenetic analysis revealed that AcdS sequence of SBP-8 is closely
related to AcdSPB and other strains belonging to genera Klebsiella,
Pseudomonas, Bacillus, and Serratia. It also showed identity with one
of the most characterized ACCD bacteria Pseudomonas putida UW4
(Fig. 2B).
3.3. Plant-growth-promoting traits of SBP-8
The clear zone observed around bacterial growth on NBRIP
medium having TCP (tricalcium phosphate) indicated phosphate solubilizing ability of isolate SBP-8. It was further
quantied as 13.71 1.15 g ml1 phosphate made bio-available
due to bacterial activity. This isolate also showed a positive
result for the production of phytohormone indole-3-acetic acid
(0.41 0.08 g ml1 ) and gibberellic acid (11.7 g ml1 ) (Table 1).
In addition, the formation of orange halo zone on the CAS-agar
plate under iron limiting conditions indicated the synthesis of
siderophore by the isolate (Suppl. Fig. 1).
The biocontrol potential of SBP-8 was evaluated by testing the antagonistic activity against various fungal and bacterial
pathogens. This isolate was observed to be inhibitory for the growth

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R.P. Singh et al. / Journal of Plant Physiology 184 (2015) 5767

61
29
29
46

K. pneumoniae strain CCFM8349

K. pneumoniae strain sebastianvr 91
K. pneumoniae strain CCFM8354
K. pneumoniae strain CCFM8360
K. pneumoniae strain CCFM8364

K. pneumoniae strain MW-W 828

62

K. pneumoniae strain SA-C4-51

75

K. pneumoniae strain ES2

39
23

99

39

K. pneumoniae strain PSB 2

K. pneumoniae strain AGR
Klebsiella sp. CCFM8386

K. pneumoniae strain CCFM8361

Klebsiella sp. XJ15
Klebsiella sp. strain SBP8
K. pneumoniae strain HKG219

99

0.01

Fig. 1. Phylogenetic tree showing relationship of Klebsiella sp. SBP-8 to closely related bacteria. PCR amplied amplicon of partial 16S rRNA gene of SBP-8 was sequenced
and used for construction of a phylogenetic tree. The 16S rRNA gene sequence of closely related species was obtained from NCBI GenBank database. The tree was obtained
using neighbor-joining method of software packages Mega version 6.0, at bootstrap value of (n = 500).

Fig. 2. Amplication and analysis of AcdS gene of Klebsiella sp. SBP-8. Panel A shows amplicon of AcdS (650 bp) of test isolate in Lane 1 and DNA ladder mix (SM0331) in Lane
2. Panel B represents dendrogram based on AcdS sequence of test isolate and other species which showed similar AcdS sequence. Other sequences of AcdS used for analysis
were retrieved from NCBI Genbank data base. Neighbor-joining method was performed using the software packages Mega version 6.0, at bootstrap value of (n = 500).

Table 1
Plant-growth-promoting traits of strain Klebsiella sp. SBP-8.
Plant-growth-promoting traits
ACCD activity (nmol of -KB mg1 Pr h1 )
IAA production (g ml1 )
Phosphate solubilization (g ml1 )
Gibberelic acid production
Siderophore index
Chitinase activity
Ammonia production

Activity
396.20 21.18
0.4100 0.084
13.715 1.156
+
+
+
+

of A. avus (9.7 0.14 mm), F. oxysporum (17.3 0.45 mm), and F.

graminearum (18.4 0.26 mm). However, it did not show antagonism against bacterial species such as B. cereus, E. carotovora, E. coli,
and S. aureus. Isolated strain was showing swimming, swarming
and twitching motility on respective media (Suppl. Fig. 2).

3.4. Physiological characterization of ACC deaminase activity

ACCD activity of isolate SBP-8 was also measured under different
salt and temperature conditions. An increase in ACCD activity was
observed with salt concentration up to 6%. Among different salt

R.P. Singh et al. / Journal of Plant Physiology 184 (2015) 5767

63

Fig. 3. ACCD activity of isolate SBP-8 in various physiological conditions (A) salt stress conditions (B) different temperatures conditions.

concentrations, highest ACCD activity of 416.90 11.20 nmol KB mg1 Pr h1 was observed in DF media supplemented with
6% of NaCl (Fig. 3A). On the contrary, enzyme activity decreased
under temperature stress condition. Optimal enzyme activity
(371.0 12.7 nmol -KB mg1 Pr h1 ) was noted at 30 C, which
followed a further decrease in activity with the increase in temperature (Fig. 3B).
Among the various carbon sources used for the enzyme
assay of SBP-8, the highest activity of ACCD was observed
with
lactose
(327.30 21.75 nmol -KB mg1 Pr h1 )
followed by dextrose (312.20 24.50 nmol -KB mg1 Pr h1 ),
sucrose (288.20 18.90 nmol -KB mg1 Pr h1 ) and xylose
(131.10 21.60 nmol -KB mg1 Pr h1 ). Similarly, varied activity
of ACCD was noted in different nitrogen sources, where the highest
activity (412.00 21.90 nmol -KB mg1 Pr h1 ) was observed with
ammonium chloride. The activity in different nitrogen sources was
in following order: NH4 Cl > NH4NO3 > (NH4 )2SO4 > KNO3 . Different
concentrations of the substrate were used for measuring enzyme
activity which indicated that 3 mM is optimal ACC concentration
for enzyme activity (396.20 21.18 nmol -KB mg1 Pr h1 ). Other
measures such as pH and incubation time were used, which
demonstrated pH 8.0 (420.00 17.70 nmol -KB mg1 Pr h1 ) and
24 h incubation time (391.30 29.60 nmol -KB mg1 Pr h1 ) were
optimal for ACCD activity.
3.5. Physiochemical characteristics of soil for pot study
Soil was analyzed for its nutritional status for the plant
growth study. The various parameters of soil were as follows:
pH 7.20, EC 0.162 ds m1 , Olsen P 34.6 mg kg1 , OC 0.2%, total N
61 mg kg1 , K 140.6 mg kg1 , Zn 0.218 mg kg1 , Cu 0.124 mg kg1 ,
Fe 2.61 mg kg1 , Mn 0.948 mg kg1 .
3.6. Effect of Klebsiella sp. SBP-8 on plant growth under salt and
temperature stress
The effect of ACCD bacteria Klebsiella sp. SBP-8 in ameliorating
the adverse effect of abiotic stressors on plant growth was tested
by growing wheat plants under varying conditions of salt and temperature stressors. In comparison to uninoculated control plants,
a signicant increase in terms of all measured parameters was
observed in SBP-8 inoculated plants at all salt concentrations used
in this study (Table 2). Data on shoot length indicate that inoculation with the isolate enhanced shoot length signicantly (p = 0.05)
in comparison with control. The maximum increase in shoot length
(47.43%) was observed at 200 mM salt stress compared to control treated with the same concentration of salt. The maximum
increase in root length (36.05%) was observed at 200 mM salt as

compared to un-inoculated control under 200 mM salt stress. Inoculation with the bacterial isolate also signicantly promoted fresh
weight (p = 0.05) in comparison with control. The maximum shoot
fresh weight (24.52%) was observed by the isolate at 200 mM salt
stress. The highest increase in dry weight (22.47%) was observed at
150 mM NaCl as compared to control plants treated with respective
salt. Bacterial inoculation signicantly increased the chlorophyll a
as compared to uninoculated plants. The highest increase in chlorophyll a was observed at 200 mM (140.74%) followed by 175 mM
(89.02%) salt stress in SBP-8 inoculated plants as compared to
their respective control. The greatest increase in chlorophyll b was
31.39%, followed by 30.27% at 150 mM and 175 mM salt stress
respectively in bacterized plants as compared to uninoculated
plants.
Inoculation with bacteria signicantly (p = 0.05) increased the
growth of wheat plant under various temperatures. The maximum
increase in shoot length was observed at 25 C (42.82%) as compared to un-inoculated control germinated at 25 C. Inoculation
improved the root length by 57.71% at 25 C, and 25% at 30 C.
Inoculation also signicantly increased (p = 0.05) the fresh weight
up to 95.87% at 35 C and 28.19% at 30 C as compared to their
respective control. Similarly, signicant (p = 0.05) increase in dry
wt. (46.20%) was observed at 35 C and 24.40% at 30 C as compared to their respective control. The increase in chlorophyll a was
94.74%, 47.14% and 45.88% at 35 C, 30 C and 25 C respectively
as compared to their control (Table 3). It also exhibited signicant
(p = 0.05) increase in chlorophyll b with 55.88% and 38.95% at 35 C
and 30 C as compared to uninoculated plants germinated at the
respective temperature.
3.7. Analysis of ionic elements
Salinity signicantly increased Na+ concentration and decreased
and Ca2+ concentrations in wheat shoots of control plants. However, inoculation with isolate SBP-8 resulted in a decrease of Na+
and an increase of K+ concentration in plant tissue as the salt
concentrations increased. Higher Na+ content and less K+ content was observed in uninoculated plants at 200 mM NaCl than
other concentrations used. When compared to uninoculated plants,
the maximum reduction in Na+ content (65%) and increase in K+
(84.21%) were observed in inoculated plants treated with 200 mM
NaCl (Table 4). In contrast, Na+ content was unaffected in inoculated plants in non-saline (0 mM) soil. Moreover, in inoculated
plants, there was an increase in K+ content with 23.21% and 18.35%
at 175 mM and 150 mM NaCl, respectively. Increased K+ uptake
by bacterial inoculation demonstrated a clear effect on potassium
transport in plants. There was no signicant change in Ca2+ in uninoculated plants under salt stress. However, with the increase in
K+

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R.P. Singh et al. / Journal of Plant Physiology 184 (2015) 5767

Table 2
Effect of bacterial isolate SBP-8 on different growth parameters of wheat plant grown under salt stress condition.
Treatment

Salt stress (mM)

Control
Control
Control
Control
SBP-8
SBP-8
SBP-8
SBP-8

0
150
175
200
0
150
175
200

SL (cm)

RL (cm)

26.57 1.59
24.05 2.45
22.40 2.72
17.29 2.15
31.78 1.76a
29.06 1.03a
26.82 1.48a
25.49 2.02a

20.08 2.11
19.89 2.69
18.60 1.71
15.34 1.20
23.15 1.35a
22.56 1.30
21.47 2.11
20.87 2.37a

FW (g)
2.56 1.07
2.20 0.99
1.81 0.48
1.59 0.57
3.10 0.88a
2.59 1.05a
2.17 1.09a
1.98 0.75a

DW (g)
0.297 0.028
0.218 0.012
0.184 0.013
0.160 0.019
0.315 0.059
0.267 0.021a
0.220 0.013a
0.190 0.021a

Chl a (mg g1 FW)

3.25 0.56
3.10 0.15
2.37 0.48
1.08 0.20
5.59 0.74a
5.08 0.22a
4.48 0.18a
2.60 0.31a

Chl b (mg g1 FW)

1.88 0.32
1.37 0.45
1.09 0.22
0.98 0.17
2.10 0.30
1.80 0.22a
1.42 0.15a
1.16 0.13a

Growth was measured at 15 days after seed germination under different salt stress conditions. SLshoot length, RLroot length, FWfresh weight, DWdry weight. The values
are mean SD (n = 15).
a
Represent signicant difference from respective control according to Duncans multiple range test (p = 0.05).
Table 3
Effect of bacterial isolate SBP-8 on different growth parameter of wheat under various temperature conditions.
Treatment
Control
Control
Control
Control
SBP-8
SBP-8
SBP-8
SBP-8

Temp stress ( C)
25
30
35
40
25
30
35
40

SL (cm)
25.87 1.31
21.80 2.53
15.25 1.30

28.37 2.63a
27.57 2.07a
21.78 1.66a

RL (cm)
18.72 2.10
16.36 1.88
11.73 1.58

19.45 1.75
20.45 1.75a
18.50 0.70a

SFW (g)
2.49 0.033
1.88 0.090
1.21 0.074

2.60 0.020
2.41 0.080a
2.37 0.092a

SDW (g)
0.270 0.019
0.209 0.027
0.158 0.017

0.291 0.021
0.260 0.016a
0.231 0.018a

Chl a (mg g-1 FW)

3.40 0.40
2.10 1.08
0.95 0.74

4.96 1.20a
3.09 0.65a
1.85 0.54a

Chl b (mg g-1 FW)

1.55. 0.70
0.95 0.42
0.68 0.39

1.68 0.50
1.32 0.27a
1.06 0.81a

Growth was measured at 15 days after seed germination under different temperature regimes. SLshoot length, RLroot length, FWfresh weight, DWdry weight. The values
are mean SD (n = 15).
a
Represent signicant different from respective control according Duncans multiple range test (p = 0.05).

salinity slight increase in Ca2+ content was observed in inoculated

plants. Higher Ca2+ content (27.27%) were observed at 200 mM NaCl
as compared to un-inoculated plants with same salt stress treatments.

3.8. Plant colonization

The colonization ability of the isolate was determined by
plate counting, microscopy and ERICPCR of inoculated bacteria
with respect to control after the experimental period as well as
microscopic evaluation. On the 15th day after seed germination,
associative bacteria were detected in a range of 1.8 103 CFU/g
of the root. Colonization of inoculated bacteria was also evident
from the micrograph (Suppl. Fig. 3) which indicated a large number
of uorescent cells on staining with acridine orange. No bacterial
cells were observed on the root surface of uninoculated plants. To
conrm an identity of colonized bacteria, genomic DNA of treated
plants was subjected to ERICPCR based nger printing. ERICPCR
prole obtained from total genomic DNA of treated plants was
identical to that of pure culture, thereby conrming the identity
of colonized bacteria.

4. Discussion
The present study reports the role of plant-growth-promoting
Klebsiella sp. SBP-8 in improving plant growth under salt and
temperature stress conditions. The selected isolate was isolated
from the rhizosphere of S. bicolor growing in the desert region of
Rajasthan. The bacterial isolate was found to decrease the accumulation of Na+ in the host plant under salt stress condition. In
addition, SBP-8 was also investigated for the presence of ACCD
activity which can protect host plant from the deleterious effect
of given stressors. To the best of our knowledge, the present study
is the rst to report the role of a strain belonging to genus Klebsiella in enhancing the plant growth under salt and temperature
stresses. However, the growth promoting effects of Klebsiella sp.
on the wheat plant under axenic condition have been observed by
Sachdev et al. (2009). In another study, nitrogen xing Klebsiella
sp. strains enhanced the growth and development of a halophyte
Salicornia bigelovii (Castellanos et al., 2003).
Rhizospheric bacteria stimulate plant growth both by direct
and indirect mechanisms under various biotic and abiotic stresses.
High salinity is a major environmental stress factor that adversely
affects plant growth, productivity and development (Allakhverdiev
et al., 2000). Since many PGPR showing ACCD activity are known
to promote plant growth under biotic and abiotic stress conditions,

Table 4
Inuence of Klebsiella sp. SBP-8 on Na+ , K+ and Ca++ ion concentrations in shoot tissue under control and salt stress.
Treatments
Control
Control + 150 mM NaCl
Control + 175 mM NaCl
Control + 200 mM NaCl
SBP-8
SBP-8 + 150 mM NaCl
SBP-8 + 175 mM NaCl
SBP-8 + 200 mM NaCl

Na+ (mg g1 DW)

1.01 0.07
1.36 0.09
1.58 0.11
1.98 0.10
1.09 0.06
1.11 0.15a
1.18 0.19a
1.20 0.23a

K+ (mg g1 DW)
1.34 0.07
1.09 0.12
1.12 0.31
0.95 0.28
1.42 0.30
1.29 0.21a
1.58 0.08a
1.75 0.17a

Values are the means of 3 replicates SD.

a
Represent signicant difference from respective control based on Duncans multiple range test (p = 0.05).

Ca++ (mg g1 DW)

1.27 0.14
1.20 0.22
1.17 0.17
1.10 0.19
1.30 0.09
1.27 0.11
1.31 0.26
1.40 0.20a

R.P. Singh et al. / Journal of Plant Physiology 184 (2015) 5767

selection of isolate in the present study was primarily based on

high ACCD activity and other plant-growth-promoting properties.
SBP-8 showed quite high ACCD activity to trigger induced systemic
tolerance which is supported by the fact that the isolate having
>20 nmol of -KB mg1 h1 can inuence plant growth promotion due to reduction of stress ethylene produced in plants under
stress conditions (Penrose and Glick, 2003). This hypothesis has
been proven in earlier studies where inoculation of halotolerant
ACCD bacteria reduced level of ethylene by 4764% in red pepper
seedlings grown under salt stress condition (Siddikee et al., 2011,
2012). Although, measurements of ethylene production in plant
roots inoculated with an ACCD containing PGPR vs. a non-ACCD
containing strain are still lacking. The given isolate was tested for
its ability to tolerate salt and temperature stress in terms of measuring its growth and ACCD activity. It was interesting to note that
growth and enzyme activity were highest at 6% salt concentration
and the increase in ACCD activity might be due to increase in AcdS
gene expression. The increase in AcdS gene expression of Enterobacter sp. and Chryseobacterium sp. was observed on increasing the salt
stress from 0 to 100 mM NaCl (Tittabutr et al., 2013). However, both
growth and enzyme activity decreased under temperature stress.
Nevertheless, ability of this isolate to tolerate temperature up to
50 C would have remarkable importance in actual farming conditions, particularly in a place like Rajasthan, which experiences high
increases in temperature during summer (Ali et al., 2009; Gehlot
et al., 2012).
Considering the importance of ACCD in amelioration of plant
stress, the effect of different physiological factors on bacterial
ACCD was assessed. Among different carbon sources used, lactose
appeared as most preferred for ACCD activity, whereas it was least
in the presence of xylose. Similar to the effects of different carbon
sources, ACCD enzyme activity was also assessed in the presence
of different nitrogen sources, which showed the highest activity
with ammonium chloride. Results of ACCD activity of SBP-8 with
relation to the concentration of substrate (ACC), pH and incubation period tallied with previous reports (Jha et al., 2012). Further,
ACCD activity was characterized at the molecular level by amplifying AcdS, a gene encoding for ACCD in bacteria. The appearance
of the expected amplicon of 650 bp and its sequence analysis conrmed presence of ACCD in SBP-8. Previous studies have also shown
successful amplication of AcdS gene in a variety of bacteria (Jha
et al., 2012; Shrivastava and Kumar, 2013). Phylogenetic analysis of AcdS gene suggested that the AcdS sequence of Klebsiella sp.
SBP-8 was similar to bacteria belonging to genus Klebsiella as well
as other bacterial genera. The similarity of AcdS sequences with
diverse species suggests that the prevalence of AcdS gene may not
be directly related with phylogeny. A Recent study by Nascimento
et al. (2014) has suggested that ACCD evolves mainly through the
vertical transfer with the occasional horizontal transfer.
In addition to ACCD activity, the isolate SBP-8 was also screened
for other important plant-growth-promoting properties. The presence of a reasonable amount of phytohormones (auxin and
gibberellin), siderophore and phosphate solubilization activity are
additional features of this isolate that can benet plant growth and
productivity. The ability of plant-growth-promoting bacteria with
the above-mentioned PGP traits is known with several experimental evidences by many workers (Karthikeyan et al., 2012; Forchetti
et al., 2007; Sachdev et al., 2009). The presence of ACCD activity
in PGPR K. pneumoniae strain Kp 342 has been reported by Iniguez
et al. (2004).
In a plant growth study under laboratory conditions, inoculated plants showed an increase in growth from 11% to 72% for
different parameters tested. The greatest increase was observed
for chlorophyll a content, which indirectly indicates improvement
in photosynthetic activity. The increase in chlorophyll content and
photosynthetic activity in response to PGPR has been demonstrated

65

in some of the earlier studies (Nadeem et al., 2006; Han and Lee,
2005). More importantly, SBP-8 was effective in decreasing plant
growth inhibition due to salt and temperature stressors. It is evident from Table 2 that an application of SBP-8 decreased growth
inhibition, even at 200 mM salt in the range of 10100%. The highest decrease in inhibition was observed for root length followed
by chlorophyll a content and shoots length. Our results are in congruence with other studies where ACCD bacteria were observed
to stimulate plant growth under salt stress conditions (Kalra et al.,
2014; Jha et al., 2011). Inoculation with ACC deaminase bacterium
P. putida UW4 has been shown to enhance growth in various physiological parameters of canola plants under an inhibitory level of
salt stress (Cheng et al., 2007). Similarly, signicant increases in
shoot length, root length and number of leaves of Limonium sinense
were also observed following inoculation of ACCD bacteria under
salt stress (Sheng et al., 2014). Since the isolate has the ability to
increase the shoot and root length under salt stress as compared
to control, it is likely that this growth behavior might be due to
ACCD activity and IAA production. It is evident from previous studies also where mutant lacking ACCD activity did not show increased
plant growth under salt or other stress conditions (Li et al., 2000;
Onofre-Lemus et al., 2009). However, role of ACCD in promoting
plant growth was in contrast to a few studies where plants inoculated with ACCD mutant and its wild type counterpart did not
show any difference in plant-growth-promoting effects (Contesto
et al., 2008). Therefore, further work is required to ensure the role of
ACCD in promoting plant growth under stress condition by raising
ACCD mutant of isolate SBP-8 and test their effect on plant growth
under identical growth conditions. In our study, apart from salt
stress, SBP-8 was also effective in providing tolerance to temperature stress to host plant. The increase in growth of inoculated plants
was also observed under temperature stress condition. There was
a signicant decrease in growth inhibition in the range of 1095%
at 35 C in inoculated plants. Higher inhibition was observed for
fresh weight followed by root length and dry weight. Under temperature stress, our results show a signicant difference in plant
growth between uninoculated and inoculated plants. The observed
difference in treated plants might be due to low electrolyte leakage
in inoculated plants, suggesting protection of membrane integrity
by bacteria (Barka et al., 2006).
As the salt level increases in the soil, Na+ exerts ionic competence diminishing the ability of ion uptake by plants. Exclusion of
Na+ and inux of K+ are the plants strategies for mitigating salt
induced stress (Shabala and Cuin, 2008). The present study showed
an increase in Na+ and decrease in K+ content with increasing salinity stress. However, bacterial inoculation signicantly decreased
the Na+ and increased K+ content, favoring K+ /Na+ ratio. This adds
an additional feature of the isolate SBP-8 in reducing the effect of
salt stress in host plants. In consonance with our results, increase
in K+ content was observed in tomato plants inoculated with ACCD
bacteria under salt stress (Mayak et al., 2004). Few other studies
have reported the role of PGPR induced regulation of Na+ import
by the high-afnity K+ transporter 1 (HKT1) for protection from
the effect of salt stress. Volatile organic compounds (VOC) of Bacillus subtilis GB03 confer salt tolerance by down- and up-regulating
HKT1 in roots and shoots respectively, and result in low Na+ accumulation throughout the plant in comparison to control (Yang et al.,
2009). Moreover, exopolysaccharides (EPSs) secreting PGPR have
the additional advantage of inhibiting the toxic effect of Na+ on the
plants by restricting the ow of Na+ in roots through binding of
Na+ to surface polysaccharides and make it less available to plants
(Ashraf et al., 2004; Geddie and Sutherland, 1993). Further work is
required to correlate these mineral element changes in response to
ACCD bacteria that might play a role in growth regulation in physiologically diverse conditions like salinity (Rodriguez-Rosales et al.,
2008).

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R.P. Singh et al. / Journal of Plant Physiology 184 (2015) 5767

The close association of plant and microbe resulting from successful colonization provides an optimum advantage for both of
the partners. Therefore, we wished to test the ability of bacteria to
colonize on the plant surface. The test isolate showed all forms of
motility which is required for chemotactic responses and colonization on plant surface (Vande Broek et al., 1998; Lugtenberg et al.,
2001). Further, colonization efciency of the isolate was examined by uorescence microscopy and ERICPCR. Bacterial cells were
visualized in inoculated plant roots using uorescent dye acridine
orange which binds to bacterial nucleic acids. This dye has been
frequently used for tracking microbial colonization using uorescent and confocal laser scanning microscope (Morris et al., 1997).
In addition to the visual conrmation, colonization was also conrmed using repetitive (ERIC) DNA-based nger printing approach
(Li et al., 2009). Typing and conrmation of DNA strains using
ERICPCR have been demonstrated in previous reports too (Syrmis
et al., 2004; Gupta et al., 2013)
5. Conclusion
Bacteria with multiple PGP traits will be helpful to increase
crop productivity under stress environments. In this study, we have
shown that bacteria with halotolerant properties are able to facilitate plant growth under abiotic stress conditions. The experiments
conrm that inoculation with ACCD bacterium Klebsiella sp. SBP-8
protects the plants against adverse effects of salt and temperature
stress. Our ndings basically focus on inoculation of bacteria that
could be able to ameliorate the salinity stress by integration of
several aspects including plant growth promotion through synthesis of plant required hormones, efcient ACCD activity to reduce
stress induced ethylene and regulation of ion transporters favoring
K+ /Na+ ratio in plants. In brief, observed results in this study indicate that bacterial isolate Klebsiella sp. SBP-8 could reduce damages
caused by high level of NaCl in the soil. Hence, the use of multifarious growth promoting bacteria with halotolerant properties hold
a great potential to be used as biofertilizer in saline soils.
Acknowledgements
We are grateful to Department of Biotechnology (File No.
BT/PR14527/AGR/21/326/2010), Govt. of India, New Delhi for the
support by providing the fund for carrying out the research work.
We sincerely thank Dr. Pankaj Sharma, Assistant professor, BITS
Pilani, India, and Prof. Sanjeev Chowdhary, Department of Humanities and Social Sciences, BITS Pilani, India for thoroughly reviewing
and correcting English language of the text.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.jplph.2015.07.
002
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