Journal of Plant Interactions

ISSN: 1742-9145 (Print) 1742-9153 (Online) Journal homepage: https://www.tandfonline.com/loi/tjpi20

Delineating Kocuria turfanensis 2M4 as a credible

PGPR: a novel IAA-producing bacteria isolated
from saline desert

Dweipayan Goswami, Shweta Pithwa, Pinakin Dhandhukia & Janki N.

Thakker

To cite this article: Dweipayan Goswami, Shweta Pithwa, Pinakin Dhandhukia & Janki
N. Thakker (2014) Delineating Kocuria�turfanensis 2M4 as a credible PGPR: a novel IAA-
producing bacteria isolated from saline desert, Journal of Plant Interactions, 9:1, 566-576, DOI:
10.1080/17429145.2013.871650

To link to this article: https://doi.org/10.1080/17429145.2013.871650

© 2013 Taylor & Francis Published online: 02 Jan 2014.

Submit your article to this journal Article views: 3814

View related articles View Crossmark data

Citing articles: 29 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=tjpi20
Journal of Plant Interactions, 2014
Vol. 9, No. 1, 566–576, http://dx.doi.org/10.1080/17429145.2013.871650

RESEARCH ARTICLE
Delineating Kocuria turfanensis 2M4 as a credible PGPR: a novel IAA-producing bacteria
isolated from saline desert
Dweipayan Goswamia, Shweta Pithwaa, Pinakin Dhandhukiab and Janki N. Thakkera*

a
Department of Biotechnology, P. D. Patel Institute of Applied Sciences, Charotar University of Science and Technology, CHARUSAT
Campus, Changa-388421, Anand (Gujarat), India; bAshok and Rita Patel Institute of Integrated Study and Research in
Biotechnology and Allied Sciences, ADIT Campus, New Vidyangar-388121, Anand (Gujarat), India
(Received 15 October 2013; accepted 30 November 2013)

Indole-3-acetic acid (IAA)-producing bacteria Kocuria turfanensis strain 2M4 was isolated from the rhizospheric soil of
halotolerant plant Suaeda fruticosa from a unique saline desert of Little Rann of Kutch, Gujarat, India. Rhizobacteria
was bright orange pigmented, gram-positive, coccoid, non-endospore forming, and aerobic in nature. 16S rRNA gene
sequence analysis showed that 2M4 isolate matched best with type strain of K. turfanensis HO-9042T. Isolate optimally
produced 38 µg ml−1 IAA when growth medium was supplemented with 600 µg ml−1 of L-tryptophan. Thin layer
chromatography and Fourier transform infrared spectroscopy analysis were performed to corroborate IAA production.
To characterize rhizobacterial isolate as a plant growth-promoting bacteria, it was tested for phosphate solubilization
where it solubilized maximum 12 µg ml−1 phosphate in presence of fructose, produced 53% siderophore units under
iron-free minimal MM9 medium and produced 1.8 µmol ml−1 ammonia in peptone water broth. Plant growth promotion
by test isolate was studied on groundnut (Arachis hypogaea L.) under non-saline and saline soil. There was increase by
18% in total plant length and 30% in fresh biomass observed under non-saline control soil. Under saline soil, test isolate
showed 17% increase in total length of the plant and 13% increase in fresh biomass.
Keywords: Kocuria turfanensis; indole-3-acetic acid (IAA); Fourier transform infrared spectroscopy (FTIR); thin layer
chromatography (TLC); Little Rann of Kutch

Introduction (1978) addressing the group of bacteria that helps to

The genus Kocuria was proposed by Stackebrandt et al.
(1995) which was earlier classified in the genus Micro-
coccus. The members of this genus are gram-positive,
coccoid, aerobic, non-encapsulated, and non-endospore
forming belonging to the order of Actinomycetales. This
genus of Actinobacteria has been isolated from different
sources such as air, fermented sea food, mammalian
skin, soil, the rhizoplane, freshwater, seawater, marine
sediment, and desert soil (Kaur et al. 2011). At present,
17 species belonging to Kocuria genus have validly
published names, available at http://www.bacterio.cict.fr/
k/kocuria.html. The genus Kocuria includes several
halotolerant strains including K. kristinae, K. rhizophila,
and K. marina which can tolerate up to 10% NaCl in
growth medium (Kovacs et al. 1999; Kim et al. 2004).
K. varians, K. rhizophila, K. flava, and other unnamed
species of Kocuria have been reported to produce large
amount of indole-3-acetic acid (IAA) (Saharan & Nehra
2011; Vicene et al. 2012). IAA is a common natural
auxin and a product of L-tryptophan metabolism of
microorganisms. It influences several parameters in the
plant, namely elongation and cell division, apical dorm-
ancy, and differentiation of vascular tissues (Aloni et al.
2006; Ali et al. 2010).
IAA production is one of the important trait of plant
growth-promoting bacteria widely known as PGPR. This
term PGPR was first coined by Kloepper and Schroth
enhance plant growth by inhabiting rhizosphere of plant.
Commonly known traits of PGPR include phytohormone
production such as IAA, production of ammonia from
nitrogenous organic matter, solubilization of phosphate,
production of siderophores which helps in suppression
of plant pathogenic bacteria, and so on (Ganeshan &
Kumar 2005; Ahmad et al. 2008; Ali et al. 2011). Most
routinely studied PGPR species producing IAA belongs
to Pseudomonas, Bacillus, Azotobacter, Azospirrulam,
and Rhizobia genus (Aeron et al. 2011; Bhattacharyya &
Jha 2012). Despite of well-documented history for
species belonging to Kocuria genus as IAA producers,
they have been poorly investigated for their potential
as PGPR.
In this present work, we isolated efficient IAA-
producing strain ‘2M4’ from the rhizosphere of Suaeda
fruticosa, native of saline desert of Little Rann of Kutch,
Gujarat, India. 2M4 isolate produced bright orange-
colored colonies on nutrient agar and could tolerate up
to 8% NaCl in the growth medium (Goswami et al.
2013a). 16S rRNA gene sequence analysis showed the
isolate belonged to Kocuria genus. Further biochemical
characterization of Kocuria sp. 2M4 matched with the
type strain of Kocuria turfanensis HO-9042T as
described by Zhou et al. (2008). Kocuria sp. 2M4 was
further tested for other plant growth-promoting traits
including phosphate solubilization under the presence of

*Corresponding author. Email: jankithakker.bt@ecchanga.ac.in

© 2013 Taylor & Francis
 Journal of Plant Interactions
various sugars, ammonia production, and siderophore
production. To test the plant growth-promoting ability of
Kocuria sp. 2M4, talc-based bioformulation was pre-
pared and its efficacy was tested on the seedling growth
of groundnut (Arachis hypogaea L.) in presence and
absence of NaCl stress under pot trials.
Materials and methods
Isolation and screening for IAA-producing bacteria
Rhizospheric soil of halotolerant plant S. fruticosa from
a unique saline desert of Little Rann of Kutch, Gujarat,
India (23°51′N, 71°28′E) was collected. Soil sample was
suspended in standard normal saline in the ratio of 1:9
and vortex for 20 min at room temperature. The
suspension of soil in normal saline was serially diluted
and its 0.1 ml aliquot was inoculated on modified
nutrient agar (peptic digest of animal tissue 5.0 g L−1,
sodium chloride 5.0 g L−1, beef extract 1.5 g L−1, yeast
extract 1.50 g L−1, and final pH at 25°C was 7.4 ± 0.2)
procured from HiMedia Laboratories, supplemented with
5% NaCl using spread plant technique and incubated for
24 h at 35 ± 2°C. Morphologically distinct colonies were
subcultured to obtain pure cultures.
To screen the halotolerant organisms producing IAA,
all the isolates obtained were grown in the nutrient broth
supplemented with 5% NaCl and 500 µg ml−1 of
L-tryptophan. Spectrophotometric estimation of IAA
was performed as per the method developed by Brick
et al. (1991) with modifications adapted by Goswami
et al. (2013b). 72-h old culture supernatants of the
bacterial isolates were mixed with Salkowski reagent (50
ml, 35% of perchloric acid, 1 ml 0.5 M FeCl3 solution)
in the ratio of 1:1. Development of pink color indicates
the production of IAA and its optical density was
recorded at 530 nm. Concentration of IAA produced
was estimated against standard curve of IAA (HiMedia)
in the range of 10–100 µg ml−1. We have already
published this part of research involving isolation and
screening of IAA-producing bacteria (Goswami et al.
2013a). Present focus is to identify, characterize, and test
plant growth-promoting ability of novel isolate desig-
nated 2M4 which showed bright orange colored colonies
on nutrient agar and produced IAA.
Identification of bacterial isolate using 16S rRNA gene
sequence
16S rRNA gene sequence analysis of IAA-producing
bacterial isolate was performed to identify the organism.
To amplify 16S rRNA gene, universal forward primer:
5′-AGAGTTTGATCCTGGCTCAG-3′ and reverse pri-
mer: 5′-AAGGAGGTGATCCAGCCGCA-3′ from 1st
BASE (Agile Lifescience Technologies India Pvt. Ltd.)
were used. Amplified gene product ∼1.6 kb was
sequenced at 1st BASE. Gene sequence homology was
tested using BLASTn search program (http://www.ncbi.
nlm.nih.gov). Gene sequence obtained was aligned by
ClustalW using MEGA 4.0 software (Tamura et al.
567
2007) and a neighbor-joining tree with bootstrap value
500 was constructed. The gene sequence was submitted
to GenBank and accession number was assigned.
Biochemical characterization
Morphological analyses were performed by using a
standardized method (Murray et al. 1994). Physiological
tests including growth at different temperatures, pH, and
NaCl concentrations were performed by growing the
strain in nutrient broth under appropriate test conditions.
Biochemical tests described in Bergey’s Manual of
Systematic Bacteriology were performed as per the
methods described by Shi et al. (2012). All the media
and reagents required to perform biochemical tests were
obtained from HiMedia. Sugar utilization tests were
performed using HiCarbo™ Kit-KB009 from HiMedia.
Characterization of bacterial IAA
Correlation between IAA produced and supplemented L-
tryptophan was determined. Isolate 2M4 was grown in
nutrient broth with varying concentrations of supple-
mented L-tryptophan in the range of 0–600 µg ml−1 and
IAA was determined at 24 h, 48 h, 72 h, 96 h, and 120 h
as per the spectrophotometric method described earlier.
To confirm the production of IAA by isolate 2M4,
IAA was extracted from the 72-h old culture supernatant
and analyzed using thin layer chromatography (TLC)
and Fourier transform infrared spectroscopy (FTIR). To
extract the IAA produced by isolate 2M4, 72-h old
culture supernatant (as sample) and un-inoculated
medium supplemented with L-tryptophan (as control)
were acidified to pH 2.5 using 1 N HCl and extracted
thrice using equal volume of ethyl acetate. Fraction of
ethyl acetate was air dried and redissolved in one-tenth
of the culture supernatant volume in methanol.
TLC analysis for confirmation of IAA production
For TLC analysis, standards of L-tryptophan, IAA, and
indole-3-butyric acid (IBA) were prepared in methanol
(200 µg ml−1), 30 µl of bacterial methanolic extract along
with each standard were spotted on aluminum-backed
silica gel 60 F254 TLC foils (4 × 6 cm) of 0.25-mm
thickness (Merck, Darmstadt, Germany). Spots were
allowed to develop using the mobile phase isopropanol:
ammonia:water (16:3:1 [v/v]). After running, TLC foils
were dried and observed under 256- and 360-nm UV light.
FTIR analysis for confirmation of IAA production
In order to confirm the production of IAA by bacterial
isolate based on information about its chemical bonds
and molecular structure, FTIR analysis was carried out.
Methanolic extract was completely dried and mixed with
spectral grade potassium bromide, and FTIR spectral
analysis of IAA was recorded at the transmission mode
from 400–4000 cm−1 using Thermo Scientific Nicolet
FTIR 6700.
568 D. Goswami et al.
Assessment of phosphate solubilization ability
The method developed by Pikovskaya (1948) was used
for quantitative estimation of tricalcium phosphate solu-
bilization. Phosphate solubilization ability of test isolate
was determined in presence of different sugars (conc.
1%) including glucose, fructose, sucrose, and mannitol
in the modified Pikovskaya’s medium. The concentration
of the soluble phosphate was estimated from the
supernatant by stannous chloride method (King 1932)
after seven days of incubation at 30 ± 2°C. Drop in the
pH caused by organic acid production due to sugar
utilized by test isolate was determined.
Ammonia production
Nesslerization reaction described by Cappucino and
Sherman (1992) was employed to estimate ammonia.
Bacterial isolate was grown in peptone water broth for
five days at 29 ± 2°C. Culture supernatant (0.2 ml) was
mixed with Nessler’s reagent (1 ml) and volume of this
mixture was made up to 8.5 ml by the addition of
ammonia-free distilled water. Development of brown to
yellow color was measured at 450 nm using spectropho-
tometer. The concentration of ammonia was estimated
using the standard curve of ammonium sulfate in the
range of 0.1–1 µmol ml−1.
Quantitative estimation siderophore
Siderophore produced by isolate was quantified using
chrome azurol S (CAS) shuttle assay described by Payne
(1994). Test isolate was grown in iron-free MM9
minimal medium (HiMedia) supplemented with 1% (w/
v) glucose. CAS assay solution was mixed with culture
supernatant in equal proportion and allowed to stand for
20 min. Presence of siderophore removes the iron from
the dye complex and causes reduction in the intensity of
blue color which is recorded at 630 nm. For the
measurements, minimal medium was used as blank and
% siderophore units were calculated by following
formula – [(Ar − As)/Ar] 100 = % siderophore units,
where Ar = absorbance of reference (minimal media +
CAS assay solution) and As = absorbance of sample.
Pot study
Talc-based bioformulation of test isolate was prepared as
per the method described by Goswami et al. (2013b).
Briefly, a loopful of bacterial culture was inoculated into
nutrient broth and incubated in a rotary shaker at 150
rpm for 48 h at 27 ± 2°C. Talc powder was taken in a
metal tray and its pH was adjusted to neutral by the
addition of 15 g of CaCO3/kg of talc followed by the
addition of 10 g CMC to 1 kg of neutralized talc, mixed
well, and autoclaved. Four hundred milliliter of 48-h
grown bacterial suspension with colony forming unit of
2.07 × 108/ml was mixed with carrier–cellulose mixture
under aseptic conditions. After drying (approximately
35% moisture content), bioformulation was stored in
sterile plastic bag. Efficacy of bioformulation was tested
on the seeds of hybrid variant Gujarat Junagadh
Groundnut-2 (GJG-2) of groundnut. The purpose of
using talc-based bioformulation was to allow bacterial
cells to adhere seed coat due to the presence of CMC
which acts as a binding agent. Second, talc-based
bioformulation improves the viability of bacterial cells
and shelf life of the biofertilizer. Pot study was carried
out in the month of February where the average
temperature was below 35°C, night temperature ranging
from 14°C to 19°C and average humidity was less than
30%. Soil used for the experiment was sterilized by
autoclaving and it was obtained from farm (22°59′N, 72°
82′E) located near CHARUSAT University, Anand,
Gujarat which can support the growth of groundnut.
Pot trials were performed in two sets of soil where first
set of soil was unmodified soil (did not contained NaCl
supplementation) whereas, second set of soil was modi-
fied by supplementation of additional 50 mM NaCl.
Electrical conductivity of soil was measured to deter-
mine innate soil salinity. Chemical properties and
salinity of the soil were determined as per the methods
described by Ibekwe et al. (2010). For pH and EC1:5
(electrical conductivity of salt solution at 1:5 soil:water
dilution) measurement method given by Walkley and
Black (1934), available phosphorus was estimated by the
method given by Olsen et al. (1954), available potassium
by the method given by Schollenberger and Simon
(1945). Estimation of magnesium and sulfate content of
the soil samples was performed using an atomic absorp-
tion spectrophotometer as described by Chapman and
Pratt (1978), all the tests were performed by Gujarat
Laboratories, Ahmedabad. Seeds of hybrid variant GJG-
2 of groundnut were used for pot experiment. Seeds
were surface sterilized by gently shaking in 70% ethanol
for 2 min followed by HgCl2 (1%) for 3 min and finally
rinsed twice by sterile distilled water before been sowed
in pots containing biofertilizer (0.5 g bioformulation per
kilograms soil). After 15 days of germination, all the
plantlets were uprooted and various vegetative para-
meters were measured. These parameters were compared
with PGPR-treated and non-treated plantlets under con-
trol and saline conditions.
Statistical analysis
Analysis of variance (ANOVA) was carried out using
triplicate value to identify significant difference in each
vegetative parameter between treated and non-treated
seeds for both control and saline conditions. Mean
values of triplicates were compared at significance levels
of 5%, 1%, and 0.1% least square difference (LSD).
Results
Isolation
Out of all the isolates obtained from the rhizosphere of S.
fruticosa on nutrient agar amended with 5% NaCl, only
single isolate designated 2M4 was able to produce IAA
 Journal of Plant Interactions 569

above 35 µg ml−1 (Goswmi et al. 2013b) 2M4 isolate
produced bright orange pigmented small, convex colon-
ies with entire margin. 2M4 was gram-positive, coccoid,
did not produce endospore, and it was aerobic in nature.
Further experiments were performed using 2M4 isolate.
Identification of 2M4 isolate using 16S rRNA gene
sequence analysis
Phylogenetic analysis was performed to identify 2M4
isolate. Phylogenetic tree was constructed by aligning
16S rRNA gene sequences of species belonging to genus
Kocuria with validly published names along with 16S
rRNA gene sequence of 2M4. Type strains belonging to
Kocuria genus used to construct phylogenetic tree were
K. himachalensis, K. rosea, K. polaris, K. aegyptia, K.
turfanensis, K. flava, K. palustris, K. kristinae, K.
carniphila, K. marina, K. varians, and K. rhizophila.
Phylogenetic analysis showed that 16S rRNA gene
sequence 2M4 matched best with gene sequence of
type strain K. turfanensis HO-9042T under GenBank ID-
DQ531634. Based on biochemical characterization and
phylogenetic analysis, 2M4 isolate was designated as K.
turfanensis strain 2M4 and its 16S rRNA gene sequence
was submitted GenBank nucleotide sequence database
under the accession number JX679496.
Biochemical characterization of 2M4
Physiological tests to study the growth of 2M4 under
various pH, salinity, and temperature showed that the
isolate can grow opulently within the pH range of 6–9;
however, optimum pH for the growth was 8. Isolate
could grow best when 2% (w/v) NaCl was supplemented
in nutrient broth and grew up to 6% NaCl supplementa-
tion (Figure 2). 2M4 can survive being dormant up to
10% NaCl supplementation. 2M4 isolate grew optimally
at 29 ± 2°C. Growth was not observed above 40°C and
below 22°C.
Biochemical tests of 2M4 are shown in Table 1,
where it was found negative for amylase, gelatinase,
urease, oxidase, indole production, methyl red test, and
Voges–Proskauer test. 2M4 was found positive for
catalase, slow positive for esculin hydrolysis, and nitrate
reduction. Substrates used for growth and acid produc-
tion by 2M4 were xylose, maltose, fructose, dextrose,
trehalose, sucrose, L-arabinose, glycerol, inositol, man-
nitol, rhamnose, melezitose, and citrate. 2M4 could not
utilize lactose, galactose, raffinose, melibiose, mannose,
inulin, sodium gluconate, salicin, dulcitol, adonitol,
arabitol, erythritol, α-methyl-d-glucoside, α-methyl-d-
mannoside, xylitol, ortho-Nitrophenyl-β-galactoside
(ONPG), and sorbose.
Characterization of IAA produced by 2M4
It was found that IAA produced was L-tryptophan
dependent. Production of IAA increases with increase
in supplemented L-tryptophan concentration in the
medium. Maximum IAA produced by 2M4 isolate was
38 µg ml−1 when the growth medium was supplemented
with 600 µg ml−1 of L-tryptophan after 96 h of
incubation at 29 ± 2°C under constant agitation. IAA
production increased till 96 h of incubation, and there
after it reached plateau. However, 4 µg ml−1 of IAA was
produced when growth medium was not supplemented
with L-tryptophan (Figure 3).

Figure 1. Phylogenetic neighbor-joining tree based on 16S rRNA gene sequences showing the relationship between K. turfanensis
strain 2M4 and related members of the genus Kocuria. Bootstrap values (expressed as percentages of 500 replications) greater than
50% are given at nodes. Bar 0.5% sequence variation. GenBank accession numbers are given in parentheses.
570
Figure 2. Growth of 2M4 at 29 ± 2°C with constant aeration (a) at various pH when NaCl concentration was 0.5% in nutrient broth
and (b) at various NaCl concentrations when pH of the medium was kept neutral.
Table 1. Biochemical characterization of 2M4 isolate.
Biochemical tests
Gram reaction
Endospore formation
Sugar utilization tests:
Xylose, maltose, fructose, dextrose, trehalose,
sucrose, L-arabinose, glycerol, inositol, mannitol,
rhamnose, melezitose, citrate
Lactose, galactose, raffinose, melibiose, mannose,
inulin, sodium gluconate, salicin, dulcitol, adonitol,
Arabitol, erythritol, α-Methyl-D-glucoside,
α-Methyl-D-mannoside, xylitol, ONPG, sorbose
Esculin hydrolysis
Amylase
Gelatinase
Urease
Oxidase
Catalase
Indole production
Methyl red test
Voges–roskauer test
Nitrate reduction
Note: ‘ + ’ indicates positive test result, ‘ − ’ indicates negative test
result, and ‘(+)’ indicates slow positive test result.
TLC analysis
Development of spots on TLC foils is observed under
short and long UV light (Figure 4). TLC foil contains three
standards namely L-tryptophan, IAA, and IBA on first
three spots from left, respectively. Spots 4 and 5 were
loaded with extracted IAA from 2M4 cell-free supernatant
in methanol. Rf value of L-tryptophan, IAA, and IBA were
0.29, 0.35, and 0.44, respectively. Spots 4 and 5 has Rf
value of 0.35 were visible under short and long UV,
indicating presence of IAA. TLC analysis is a qualitative
test to determine the presence of IAA with greater surety
than traditional spectrophotometric method.
FTIR analysis
Characterization of IAA based on FTIR spectra was
performed as per Kamnev et al. (2001). Figure 5 shows
D. Goswami et al.
the FTIR spectra of extracted IAA from 2M4. Charac-
teristic (N–H) stretching of indole moiety is observed at
Traits 3389 cm−1 (N–H) bending, and wagging was observed
+ at 1652 cm−1 and 517 cm−1, respectively. Stretching for
− (C–N) bond of indole ring was observed at 1247 cm−1.
+ For aromatic ring, (C–H) stretching and banding was
observed at 3055 cm−1 and 740 cm−1, respectively.
Furthermore, (C = C) banding and in-ring (C–C) stretch-
− ing was observed at 1558 cm−1 and 1488 cm−1,
respectively. Alkyl (–CH2) asymmetric stretching, sym-
metric stretching, and bending was observed at 2940
cm−1, 2925 cm−1, and 1457 cm−1, respectively. Strong
(+)
−
bond of (C = O) in IAA appears at 1701 cm−1. FTIR
− spectra confirm the presence of IAA.
−
−
+
− Phosphate solubilization by 2M4
− Phosphate solubilization ability of 2M4 was determined
− in Pikovskaya’s medium along with modifications where
(+)
glucose was replaced by fructose, sucrose, and mannitol.
Tri-calcium phosphate solubilized by 2M4 isolate in
presence of different sugars is described Figure 6.
Maximum phosphate solubilized was 12 µg ml−1 in
presence of fructose, followed by 9 µg ml−1 in presence
of sucrose. However, in presence of glucose and
mannitol phosphate solubilized was reduced to 3.5 µg
ml−1 and 5.5 µg ml−1, respectively. It was observed that
pH of the medium was more acidic in the presence of
sugars which solubilized phosphate in greater amounts
(Figure 6).
Ammonia and siderophore production
It was observed that 2M4 isolate produced ammonia in
peptone water and produced siderophore in iron-free
MM9 minimal medium supplemented with 1% glucose.
Maximum production of ammonia and siderophores was
1.8 µmol ml−1 and 53% units, respectively, after 72 h of
incubation at 30 ± 2°C with constant agitation. However,
ammonia and siderophore produced in the respective
 Journal of Plant Interactions 571

Figure 3. Co-relationship between IAA produced by K. turfanensis strain 2M4 after various hours of incubation in presence of
different concentrations of supplemented L-tryptophan in the growth medium (nutrient broth).

Figure 4. TLC analysis of extracted IAA from the growth medium of K. turfanensis strain 2M4. Spots 1, 2, and 3 are standards
namely, L-tryptophan, IAA, and IBA, respectively. Spots 4 and 5 are duplicates containing extracted IAA from K. turfanensis strain
2M4. (a) and (b) shows the visualization of TLC foil under short UV (256 nm) and long UV (366 nm), respectively.

growth medium did not increase after 72 h of incubation
(Figure 7).
Pot study
As 2M4 isolate showed multiple traits of PGPR includ-
ing production of IAA, ammonia, siderophore, and
solubilization of phosphate its efficacy as a PGPR was
tested in vivo for the test plant of groundnut under non-
saline and saline conditions. Chemical properties of soil
used for pot study are shown in Table 2. Electrical
conductivity (EC1:5) of natural unmodified soil was
found to be 0.21 mS cm−1 which indicates inherent
salinity of soil to be equivalent to 32 mM NaCl and after
modification by the addition of 50 mM NaCl to these
soil EC1:5 increased to 0.64 mS cm−1 which indicates
inherent salinity of soil to be equivalent to 82 mM NaCl.
EC is indirect measure to determine the salinity of soil.
Comparing the vegetative parameters of control
plants (PGPR non-treated) under non-saline and saline
soil, it was found that total length of the plant was
reduced by 42% and fresh biomass was reduced by 43%
for plant grown under saline conditions. Test plant
(PGPR treated) grown in absence of NaCl showed
significant enhancement in the growth where there was
an increase by 18% in total plant length, 26% in root
length, 30% in fresh biomass, and 31% in dry biomass.
Test plant (PGPR treated) in presence of supplemented
50 mM NaCl showed 17% increase in total length of the
plant, root length increased by 19%, fresh biomass by
13%, and dry biomass by 17%. Table 3 shows the
comparative analysis of various vegetative parameters of
572 D. Goswami et al.

Figure 5. FTIR spectra of extracted IAA from K. turfanensis strain 2M4.

Figure 6. Phosphate solubilization and drop in pH due to acid production by K. turfanensis strain 2M4 in presence of various carbon
sources replaced in Pikovskaya’s medium.

control and test plants grown in non-saline and sa-
line soil.
Discussion
Isolation source of K. turfanensis strain 2M4 was S.
fruticosa’s rhizosphere, a plant belonging to Little desert
of Kutch. This is the unique saline desert with salinity up
to 12% (w/w) where majority of land in this desert
contain 60% clay, unlike other deserts which contains
high sand content (Gupta & Ansari 2012). Little Rann of
Kutch is nominated to be a ‘biosphere reserve’ by
UNESCO’s Man and Biosphere program (UNESCO
World Heritage Centre). Under present study attempt
was made to isolate unique rhizobacteria, as this region
is least explored for isolating rhizobacteria from the
plants adapted to its unique saline ecosystem.
To characterize any rhizobacteria as PGPR it should
be attributed to the production of diverse metabolites
including IAA, siderophores, ammonia, and activities
 Journal of Plant Interactions
Figure 7. Production of ammonia in peptone water broth and siderophore production in iron free MM9 medium supplemented with
1% glucose.
such as phosphate solubilization (Glick 1995; El-Deeb
et al. 2012). IAA production is the most prominent trait
observed by K. turfanensis strain 2M4. IAA is the
phytohormone which influences several parameters in
plant, namely elongation and cell division, apical dorm-
ancy, and differentiation of vascular tissues (Kloepper &
Schroth 1978). Godinho et al. (2010) have reported that
Kocuria rosea isolated from sand dunes of Goa pro-
duced IAA 26 µg ml−1, Vicene et al. (2012) reported
unnamed Kocuria sp. PWN-228A isolated from Pine-
wood pinaster produced 35 µg ml−1. This shows that
few species of Kocuria has been reported for IAA
production. Ahmad et al. (2008) reported that several
species of Azotobacter, Pseudomonas, and Bacillus
produced IAA up to 20 µg ml−1, 25 µg ml−1, and 10
µg ml−1, respectively. Under present study, it can be
construed that K. turfanensis strain 2M4 can produce
greater amount IAA when compared with several well-
known PGPR.
IAA is a product of L-tryptophan metabolism of
microorganisms. So for several PGPR, IAA production
is directly dependent on availability of L-tryptophan
(Ahmad et al. 2008). Under present study, K. turfanensis
strain 2M4 showed increased IAA production when
concentration of supplemented L-tryptophan was
increased. However, IAA is not the only metabolite
produced by L-tryptophan metabolism. IBA, indole-3-
pyruvate, indole-3-acetamide, tryptamine, and indole-3-
acetonitrile can be produced other than IAA by L-
tryptophan metabolism (Godinho et al. 2010). Further-
more, there have been reports where these L-tryptohan
metabolites react with Salkovaski reagent and give color
reaction (Glickmann & Dessaux 1995; Szkop & Bie-
lawski 2013). So to confirm IAA production by K.
turfanensis strain 2M4, TLC, and FTIR were performed.
Moreover, it has been reported that concentration of IAA
produced tends to be greater than other metabolites
produced by L-tryptophan metabolism (Glickmann &
Dessaux 1995; Kang et al. 2006).
Other than IAA production K. turfanensis strain 2M4
was also found to solubilize phosphate, produce side-
rophore, and produce ammonia from peptone. Phosphate
573
and nitrogen are the two main nutrients required by
plant. Organic acid secretion by rhizobacteria has been
major factor responsible for phosphate solubilization
(Buch et al. 2010). Rhizobacterial population on the
root surface is 10 times higher than in bulk soil. These
rhizobacteria utilizes sugars mainly glucose, fructose,
sucrose, xylose, and L-arabinose which are present in
root exudates metabolize it and produce organic acid
which aid in solubilization of phosphate (Archana et al.
2012). Surprisingly, biochemical analysis of K. turfa-
nensis strain 2M4 showed that it can utilize all these
important sugars to produce organic acid. However,
phosphate solubilization assays showed that maximum
solubilization of phosphate occurred in presence of
fructose and pH of the medium was more acidic as
compared to other sugars indicating greater organic acid
production in presence of fructose. Ammonia production
is another important trait of PGPR where an organism
can break down complex nitrogenous materials like
peptones and release ammonia in soil. Released ammo-
nia is taken up by plant as a nutrient source. Soils which
are rich in nitrogen, there can be an accumulation of
ammonia creating alkaline condition of the soil which
suppresses the growth of certain fungi (Jha et al. 2012).
Under present study, ammonia produced by K. turfanen-
sis strain 2M4 is at parity with several PGPR strains
including Bacillus, Pseudomonas, Rhizobium, Azotobac-
ter, and Enterobacter (Joseph et al. 2007; Jha
et al. 2012).
Siderophores are the compounds which can chelate
iron and cause iron deficiency on growing pathogens in
the plant rhizosphere, indirectly suppressing the growth
of these pathogens. The importance of siderophore
production in species of Pseudomonas is vividly
described by Jan et al. (2011), where it induced systemic
resistance in watermelon against gummy stem rot while
the siderophore-negative mutants failed to induce resist-
ance. Haas and Défago (2005) reported that strains of
Pseudomonas act as a strong biocontrol PGPR because
of its efficacy to produce siderophores. They further
reported that strains of Pseudomonas can suppress
Fusarium oxysporum, Gaeumannomyces graminis,
 574
Table 2. Analysis of chemical properties of soil.

EC1:5 of
Sodium Potassium Phosphate as P2O5 SO4 (mg Magnesium unmodified Soil salinity of EC1:5 after 50 mM NaCl Total salinity after 50-mM
pH (as Na+) (as K+) Nitrogen (mg Kg−1 of soil) kg−1 of soil) (as Mg+) soil unmodified soil supplementation NaCl supplementation

6.8 0.038% 0.37% 0.051% 14.6% 0.32% 0.006% 0.21 mS cm−1 Equivalent to 0.64 mS cm−1 Equivalent to
32-mM NaCl 82-mM NaCl

D. Goswami et al.
Table 3. Effect of 2M4-based bioformulation treatment on the seedling growth of groundnut after 15 days after been sown in pots with and without NaCl supplementation. Values are the mean of
triplicates with standard error of mean.

Soil not supplemented with NaCl Supplemented 50-mM NaCl in soil

Vegetative parameters Control 2M4 treatment p Value Significance Control 2M4 treatment p Value Significance
* *
Total plant length (cm) 26.6 ± 1.87 31.5 ± 1.21 0.0191 15.4 ± 0.98 19.06 ± 1.05 0.0115
Stem length (cm) 15.37 ± 1.48 17.3 ± 1.93 0.2429 ns 8.1 ± 0.75 10.36 ± 1.41 0.0710 ns
**
Root length (cm) 11.22 ± 0.39 14.2 ± 0.96 0.0077 7.3 ± 0.4 8.7 ± 1.13 0.1143 ns
** *
Root fresh mass (mg) 439.66 ± 10.01 503.33 ± 18.92 0.0067 318 ± 19.28 352.0 ± 6.55 0.0445
*
Root dry mass (mg) 39.66 ± 2.08 49.33 ± 5.50 0.0466 38.33 ± 6.80 45.33 ± 2.88 0.1763 ns
*** *
Stem fresh mass (mg) 1393.67 ± 81.36 1880 ± 87.17 0.0021 724.33 ± 59.21 835 ± 20.51 0.0377
* *
Stem dry mass (mg) 215.66 ± 15.56 296.33 ± 46.75 0.0471 133.33 ± 11.93 156 ± 6.24 0.0434
** *
Fresh biomass (mg) 1833.33 ± 85.04 2383.33 ± 106.10 0.0021 1042.33 ± 52.53 1187 ± 27.05 0.0132
** *
Dry biomass (mg) 255.33 ± 15.01 336 ± 38.62 0.0021 171.66 ± 8.02 201.33 ± 8.50 0.0117
Note: p Value has been calculated using one-way ANOVA and its interpretation is as follows:
ns (p Value greater than 0.05) nonsignificant as compared to control.
* (p Value between 0.05 and 0.01) significant at 5%.
** (p Value between 0.01 and 0.001) significant at 1% as compared to control.
*** (p Value less than 0.001) significant at 0.1% as compared to control.
 Journal of Plant Interactions
Rhizoctonia solani, and so on. Siderophore production
trait was observed in K. turfanensis strain 2M4 suggests
that this strain may aid in suppressing phytopathogen in
the rhizosphere.
Several published reports suggest that if an organism
shows in vitro PGPR traits such as IAA production,
phosphate solubilization, ammonia production, and side-
rophore production then it is inevitable for that organism
to shows plant growth promotion in vivo (El-Deeb et al.
2012, 2013; George et al. 2013). Deepa et al. (2010)
showed that strains of Enterobacter aerogens and
Enterobacter cloacae which produced IAA, sidero-
phores, hydrogen cyanide (HCN), and solubilized
showed growth promotion in cowpea (Vigna unguiculata
L.). Similarly, Jha et al. (2012) showed the growth
promotion in Jatropha curcas by multi-trait PGPR with
ACC deaminase activity Enterobacter cancerogenus
MSA2. Goswami et al. (2013b) reported growth promo-
tion in green gram (Vigna radiata) and chickpea (Cicer
arietinum L.) by Pseudomonas spp. strain OG isolated
from marine water. Madhaiyan et al. (2006) reported
enhancement in growth of groundnut (Arachis hypogaea
L.) by Methylobacterium spp. which also showed
biocontrol activity against its fungal pathogen Aspergil-
lus niger and Sclerotium rolfsii. Under present study, K.
turfanensis strain 2M4 possessing important PGPR traits
showed growth promotion in groundnut (Arachis hypo-
gaea L.) under saline soil. Thus, from the evidences
obtained K. turfanensis strain 2M4 isolated from saline
desert can be considered as a PGPR.
Acknowledgments
Authors are thankful to Department of Science and Technology
(DST), New Delhi, India for the fellowship and financial aid.
Authors are also thankful to Dr. K. C. Patel R&D Centre,
Charotar University of Science and Technology (CHARUSAT)
for providing necessary facilities.
References
Aeron A, Kumar S, Pandey P, Maheshwari DK. 2011.
Emerging role of plant growth promoting rhizobacteria
in agrobiology. In: Maheshwari DK, editor. Bacteria in
agrobiology: crop ecosystems. Berlin: Springer Heidel-
berg; p. 1–36.
Ahmad F, Ahmad I, Khan MS. 2008. Screening of free living
rhizospheric bacteria for their multiple plant growth
promoting activities. Microbiol Res. 263:173–181.
Ali B, Sabri AN, Hasnain S. 2010. Rhizobacterial potential to
alter auxin content and growth of Vigna radiata (L.).
World J Microbiol Biotechnol. 26:1379–1384.
Ali SZ, Sandhya V, Grover M, Linga VR, Bandi V. 2011.
Effect of inoculation with a thermotolerant plant growth
promoting Pseudomonas putida strain AKMP7 on growth
of wheat (Triticum spp.) under heat stress. J Plant Interact.
6:239–246.
Aloni R, Aloni E, Langhans M, Ulrich CI. 2006. Role of
cytokinin and auxin in shaping root architecture: regulat-
ing vascular differentiation, lateral root initiation, root
apical dominance and root gravitropism. Ann Bot.
97:883–893.
Archana G, Buch A, Kumar GN. 2012. Pivotal role of organic
acid secretion by rhizobacteria in plant growth promotion.
575
In: Satyanarayana T, Johri BN, editors. Microorganisms in
sustainable agriculture and biotechnology. Netherlands:
Springer; p. 35–53.
Bhattacharyya PN, Jha DK. 2012. Plant growth-promoting
rhizobacteria (PGPR): emergence in agriculture. World J
Microbiol Biotechnol. 28:1327–1350.
Brick JM, Bostock RM, Silverstone SE. 1991. Rapid in-situ
assay for indole acetic acid production by bacteria
immobilized on nitrocellulose membrane. Appl Environ
Microbiol. 57:535–538.
Buch A, Archana G, Naresh Kumar G. 2010. Heterologous
expression of phosphoenolpyruvate carboxylase
enhances the phosphate solubilizing ability of fluorescent
pseudomonads by altering the glucose catabolism
to improve biomass yield. Bioresour Technol. 101:
679–687.
Cappucino JC, Sherman N. 1992. Microbiology: a laboratory
manual. 3rd ed. New York: Benjamin/Cumming.
Chapman HD, Pratt PF. 1978. Methods of analysis for soils,
plants and waters. Berkeley: Division of Agricultural
Sciences, University of California; p. 3034.
Deepa CK, Dastager SG, Pandey A. 2010. Isolation and
characterization of plant growth promoting bacteria from
non-rhizospheric soil and their effect on cowpea (Vigna
unguiculata (L.) Walp.) seedling growth. World J Micro-
biol Biotechnol. 26:1233–1240.
El-Deeb B, Bazaid S, Gherbawy Y, Elhariry H. 2012. Charac-
terization of endophytic bacteria associated with rose plant
(Rosa damascena trigintipeta) during flowering stage and
their plant growth promoting traits. J Plant Interact.
7:248–253.
El-Deeb B, Fayez K, Gherbawy Y. 2013. Isolation and
characterization of endophytic bacteria from Plectranthus
tenuiflorus medicinal plant in Saudi Arabia desert and
their antimicrobial activities. J Plant Interact. 8:56–64.
Ganeshan G, Kumar MA. 2005. Pseudomonas fluorescens, a
potential bacterial antagonist to control plant diseases.
J Plant Interact. 1:123–134.
George P, Gupta A, Gopal M, Thomas L, Thomas GV. 2013.
Multifarious beneficial traits and plant growth promoting
potential of Serratia marcescens KiSII and Enterobacter
sp. RNF 267 isolated from the rhizosphere of coconut
palms (Cocos nucifera L.). World J Microbiol Biotechnol.
29:109–117.
Glick BR. 1995. The enhancement of plant-growth by free-
living bacteria. Can J Microbiol. 41:109–117.
Glickmann E, Dessaux Y. 1995. A critical examination of the
specificity of the salkowski reagent for indolic compounds
produced by phytopathogenic bacteria. Appl Environ
Microbiol. 61:793–796.
Godinho A, Ramesh R, Bhosle S. 2010. Bacteria from sand
dunes of Goa promoting growth in Eggplant. World J
Agric Sci. 6:555–564.
Goswami D, Dhandhukia P, Patel P, Thakker JN. 2013a.
Screening of PGPR from saline desert of Kutch: growth
promotion in Arachis hypogea by Bacillus licheniformis
A2. Microbiol Res. doi:10.1016/j.micres.2013.07.004
Goswami D, Vaghela H, Parmar S, Dhandhukia P, Thakker J.
2013b. Plant growth promoting potentials of Pseudomo-
nas spp. strain OG isolated from marine water. J Plant
Interact. 8:281–290.
Gupta V, Ansari AA. 2012. Geomorphic portrait of the Little
Rann of Kutch. Arab J Geosci. doi:10.1007/s12517-012-
0743-y.
Haas D, Défago G. 2005. Biological control of soil-borne
pathogens by Fluorescent Pseudomonas. Nat Rev Micro-
biol. 3:307–319.
Ibekwe AM, Poss JA, Grattan SR, Grieve CM, Suarez D. 2010.
Bacterial diversity in cucumber Cucumis sativus rhizo-
sphere in response to salinity, soil pH, and boron. Soil
Biol Biochem. 42:567–575.
576 D. Goswami et al.
Jan AT, Azam M, Ali A, Haq Q. 2011. Novel approaches of
beneficial Pseudomonas in mitigation of plant diseases –
an appraisal. J Plant Interact. 6:195–205.
Jha CK, Patel B, Sarf M. 2012. Stimulation of the growth of
Jatropha curcas by the plant growth bacterium Enterobacter
cancerogenus MSA2. World J Microbiol Biotechnol.
28:891–899.
Joseph B, Patra RR, Lawerence R. 2007. Characterization of
plant growth promoting rhizobacteria with chickpea
(Cicer arietinum L.). Int J Plant Prod. 1:141–151.
Kamnev AA, Shchelochkov AG, Perfiliev YD, Tarantilis PA,
Polissiou MG. 2001. Spectroscopic investigation of
indole-3-acetic acid interaction with iron (III). J Mol
Struct. 563:565–572.
Kang BR, Yang KY, Cho BH, Han TH, Kim IS, Lee MC, Kim
YC. 2006. Production of indole-3-acetic acid in the plant-
beneficial strain Pseudomonas chlororaphis O6 is nega-
tively regulated by the global sensor kinase GacS. Curr
Microbiol. 52:473–476.
Kaur C, Kaur I, Raichand R, Bora TC, Mayilraj S. 2011.
Description of a novel actinobacterium Kocuria assamen-
sis sp. nov., isolated from a water sample collected from
the river Brahmaputra, Assam, India. A Van Leeuw
J Microb. 99:721–726.
Kim SB, Nedashkovskaya OI, Mikhailov VV, Han SK, Kim
KO, Rhee MS, Bae KS. 2004. Kocuria marina sp. nov., a
novel actinobacterium isolated from marine sediment. Int
J Syst Evol Microbiol. 54:1617–1620.
King JE. 1932. The colorimetric determination of phosphorus.
Biochem J. 26:292–297.
Kloepper JW, Schroth MN. 1978. Plant growth-promoting
rhizobacteria on radishes. In: Gilbert-Clarey T, editor.
Proceedings of the 4th international conference on plant
pathogenic bacteria; p. 879–882.
Kovacs G, Burghardt J, Pradella S, Schumann P, Stackebrandt
E, Marialigeti K. 1999. Kocuria palustris sp. nov. and
Kocuria rhizophila sp. nov., isolated from the rhizoplane
of the narrow-leaved cattail (Typha angustifolia). Int J
Syst Bacteriol. 49:167–173.
Madhaiyan M, Reddy BS, Anandham R, Senthilkumar M,
Poonguzhali S, Sundaram SP, Sa T. 2006. Plant growth–
promoting Methylobacterium induces defense responses
in groundnut (Arachis hypogaea L.) compared with rot
pathogens. Curr Microbiol. 53:270–276.
Murray RGE, Doetsch RN, Robinow F. 1994. Determinative
and cytological light microscopy. In: Gerhardt P, Murray
RGE, Wood WA, Krieg NR, editors. Methods for general
and molecular bacteriology. Washington, DC: American
Society for Microbiology; p. 21–41.
Olsen SR, Cole CV, Watanabe FS, Dean LA. 1954. Estimation
of available phosphorus in soils by extraction with sodium
bicarbonate. US Department of Agriculture Circular No.
939. Washington, DC: US Government Printing Office.
Payne SM. 1994. Detection, isolation and characterization of
siderophores. Method Enzymol. 235:329–344.
Pikovskaya RI. 1948. Mobilization of phosphorus in soil in
connection with the vital activity of some microbial
species. Mikrobiologiya. 17:362–370.
Saharan BS, Nehra V. 2011. Plant growth promoting rhizobac-
teria: a critical review. Life Sci Med Res. 21:1–30.
Schollenberger CJ, Simon RH. 1945. Determination of
exchange capacity and exchangeable bases in soil-ammo-
nium acetate method. Soil Sci. 59:13–24.
Shi W, Takano T, Liu S. 2012. Anditalea andensis gen. nov.,
sp. nov., an alkaliphilic, halotolerant bacterium isolated
from extreme alkali–saline soil. A Van Leeuw J Microb.
102:703–710.
Stackebrandt E, Koch C, Gvozdiak O, Schumann P. 1995.
Taxonomic dissection of the genus Micrococcus: Kocuria
gen. nov., Nesterenkonia gen. nov., Kytococcus gen. nov.,
Dermacoccus gen. nov., and Micrococcus Cohn 1872 gen.
emend. Int J Syst Bacteriol. 45:682–692.
Szkop M, Bielawski W. 2013. A simple method for simultan-
eous RP-HPLC determination of indolic compounds
related to bacterial biosynthesis of indole-3-acetic acid.
A Van Leeuw J Microb. 103:683–691.
Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA4:
molecular evolutionary genetics analysis (MEGA) soft-
ware version 4.0. Mol Biol Evol. 24:1596–1599.
UNESCO World Heritage Centre. Nomination entry. http://
whc.unesco.org/en/tentativelists/2105/
Vicene CS, Nascimento F, Espada M, Barbosa P, Mota M,
Glick BR, Oliveira S. 2012. Characterization of bacteria
associated with pinewood nematode Bursaphelenchus
xylophilus. PLoS ONE. 7:e46661. doi:10.1371/journal.
pone.004666
Walkley A, Black IA. 1934. An examination of the Degtjareff
method for determining soil organic matter and a pro-
posed modification of the chromic acid titration method.
Soil Sci. 34:29–38.
Zhou G, Luo X, Tang Y, Zhang L, Yang Q, Qiu Y, Fang C.
2008. Kocuria flava sp. nov. and Kocuria turfanensis sp.
nov., airborne actinobacteria isolated from Xinjiang,
China. Int J Syst Evol Microbiol. 58:1304–1307.