Isolation, characterization, and use for plant growth promotion under salt stress, of ACC deaminase-producing halotolerant bacteria derived from coastal soil.
ABSTRACT In total, 140 halotolerant bacterial strains were isolated from both the soil of barren fields and the rhizosphere of six naturally growing halophytic plants in the vicinity of the Yellow Sea, near the city of Incheon in the Republic of Korea. All of these strains were characterized for multiple plant growth promoting traits, such as the production of indole acetic acid (IAA), nitrogen fixation, phosphorus (P) and zinc (Zn) solubilization, thiosulfate (S2O3) oxidation, the production of ammonia (NH3), and the production of extracellular hydrolytic enzymes such as protease, chitinase, pectinase, cellulase, and lipase under in vitro conditions. From the original 140 strains tested, on the basis of the latter tests for plant growth promotional activity, 36 were selected for further examination. These 36 halotolerant bacterial strains were then tested for 1- aminocyclopropane-1-carboxylic acid (ACC) deaminase activity. Twenty-five of these were found to be positive, and to be exhibiting significantly varying levels of activity. 16S rRNA gene sequencing analyses of the 36 halotolerant strains showed that they belong to 10 different bacterial genera: Bacillus, Brevibacterium, Planococcus, Zhihengliuella, Halomonas, Exiguobacterium, Oceanimonas, Corynebacterium, Arthrobacter, and Micrococcus. Inoculation of the 14 halotolerant bacterial strains to ameliorate salt stress (150 mM NaCl) in canola plants produced an increase in root length of between 5.2% and 47.8%, and dry weight of between 16.2% and 43%, in comparison with the uninoculated positive controls. In particular, three of the bacteria, Brevibacterium epidermidis RS15, Micrococcus yunnanensis RS222, and Bacillus aryabhattai RS341, all showed more than 40% increase in root elongation and dry weight when compared with uninoculated saltstressed canola seedlings. These results indicate that certain halotolerant bacteria, isolated from coastal soils, have a real potential to enhance plant growth under saline stress, through the reduction of ethylene production via ACC deaminase activity.
- SourceAvailable from: Marco Luna-Guido[Show abstract] [Hide abstract]
ABSTRACT: Agricultural practices affect the bacterial community structure in soil. It was hypothesized that agricultural practices would also affect the bacteria involved in the degradation of crop residue. Soil was sampled from four different agricultural practices, i.e. conventional agriculture on the flat or on beds, or conservation agriculture on the flat or on beds. Cultivating crops on the flat is done traditionally, but cultivating crops on beds was introduced so as to avoid water logging during the rainy season and its potential negative effect on yields. Soil from these four treatments was amended in the laboratory with maize residue (Zea mays L.) or its neutral detergent fibre (NDF) fraction, mostly consisting of (hemi) cellulose, and incubated aerobically for 14 days. Maize residue was applied to soil as it is left in the field in conservation agriculture and NDF was added to study which bacteria were favoured by application of (hemi) cellulose. Soil was incubated aerobically while the carbon mineralization and the bacterial population were monitored. On the one hand, the relative abundance of phylotypes belonging to bacterial groups that preferred low nutrient environments was higher in soil with conservation agriculture (e.g. Acidobacteria 17.6%, Planctomycetes 1.7% and Verrucomicrobia 1.5%) compared to conventional practices (Acidobacteria 11.8%, Planctomycetes 0.9% and Verrucomicrobia 0.4%). On the other hand, the relative abundance of phylotypes belonging to bacterial groups that preferred nutrient rich environments, such as Actinobacteria, showed an opposite trend. It was 11.9% in conservation agriculture and 16.2% in conventional practices. The relative abundance of Arthrobacter (Actinobacteria) and Bacillales more than doubled when maize residue was applied to soil compared to the unamended soil and that of Actinomycetales when maize or NDF was applied. Application of organic material reduced the relative abundance of a wide range of bacterial groups, e.g. Acidobacteria, Bacteroidetes, Planctomycetes and Verrucomicrobia. It was found that application of organic material favoured the same bacterial groups that were more abundant in the soil cultivated conventionally while it reduced those that were favoured in conservation agricultureApplied Soil Ecology 06/2015; 90. · 2.21 Impact Factor
- Korean J. Soil Sci. Fert. 08/2014;
- Pakistan Journal of Botany 01/2013; 45(6):1955. · 1.21 Impact Factor
J. Microbiol. Biotechnol. (2010), 20(11), 1577–1584
First published online 15 September 2010
Isolation, Characterization, and Use for Plant Growth Promotion Under
Salt Stress, of ACC Deaminase-Producing Halotolerant Bacteria Derived
from Coastal Soil
Siddikee, M. A.1, P. S. Chauhan1, R. Anandham2, Gwang-Hyun Han1, and Tongmin Sa1*
1Department of Agricultural Chemistry, Chungbuk National University, Cheongju 361-763, Korea
2Department of Agricultural Microbiology, Agricultural College and Research Institute, Tamil Nadu Agricultural University,
Republic of India
Received: July 7, 2010 / Revised: July 28, 2010 / Accepted: July 29, 2010
In total, 140 halotolerant bacterial strains were isolated
from both the soil of barren fields and the rhizosphere of
six naturally growing halophytic plants in the vicinity of
the Yellow Sea, near the city of Incheon in the Republic of
Korea. All of these strains were characterized for multiple
plant growth promoting traits, such as the production of
indole acetic acid (IAA), nitrogen fixation, phosphorus (P)
and zinc (Zn) solubilization, thiosulfate (S2O3) oxidation,
the production of ammonia (NH3), and the production
of extracellular hydrolytic enzymes such as protease,
chitinase, pectinase, cellulase, and lipase under in vitro
conditions. From the original 140 strains tested, on the
basis of the latter tests for plant growth promotional
activity, 36 were selected for further examination. These
36 halotolerant bacterial strains were then tested for 1-
aminocyclopropane-1-carboxylic acid (ACC) deaminase
activity. Twenty-five of these were found to be positive,
and to be exhibiting significantly varying levels of activity.
16S rRNA gene sequencing analyses of the 36 halotolerant
strains showed that they belong to 10 different bacterial
genera: Bacillus, Brevibacterium, Planococcus, Zhihengliuella,
Halomonas, Exiguobacterium, Oceanimonas, Corynebacterium,
Arthrobacter, and Micrococcus. Inoculation of the 14
halotolerant bacterial strains to ameliorate salt stress
(150 mM NaCl) in canola plants produced an increase in
root length of between 5.2% and 47.8%, and dry weight
of between 16.2% and 43%, in comparison with the
uninoculated positive controls. In particular, three of the
bacteria, Brevibacterium epidermidis RS15, Micrococcus
yunnanensis RS222, and Bacillus aryabhattai RS341, all
showed more than 40% increase in root elongation and
dry weight when compared with uninoculated salt-
stressed canola seedlings. These results indicate that
certain halotolerant bacteria, isolated from coastal soils,
have a real potential to enhance plant growth under saline
stress, through the reduction of ethylene production via
ACC deaminase activity.
Keywords: ACC deaminase, plant growth promoting
rhizobacteria, halotolerant bacteria, root elongation, canola,
Plant growth promoting rhizobacteria (PGPR) comprise a
group of beneficial bacteria that can be found in the
rhizoplane and rhizosphere, the phyllosphere, or the inside
of plant tissues as endophytes . Different ecological
niches have been explored for the isolation and
characterization of PGPR and include the rhizosphere soil
of different crop plants [18, 19], arable saline soil [32, 42],
polluted or contaminated soils , composted municipal
solid waste , milk , cow dung , and insect gut
. PGPR are able to promote plant growth via direct or
indirect mechanisms, or a combination of both [15, 16].
Indirect mechanisms include the suppression of pathogens
through the action of siderophores, and the production of
antibiotics and extracellular hydrolytic enzymes [16, 40].
Direct mechanisms include an altered nutrition through the
provision of fixed nitrogen; iron through siderophores;
soluble phosphate (P) and zinc (Zn) [18, 19, 22]; the
production of phytohormones such as indole acetic acid
(IAA), cytokinin, and gibberellins [23, 31]; or by the
activity of 1-aminocyclopropane-1-carboxylic acid (ACC)
deaminase, an enzyme that can lower plant ethylene levels
that are typically increased by a wide variety of
environmental stresses such as flooding, drought, heavy
metals, organic contaminants, pathogen attacks, and salt
stress [5, 8, 17, 20, 32, 36, 45].
Salinity is a natural feature of ecosystems in arid and
semiarid regions and can also be induced by anthropogenic
Phone: +82-43-261-2561; Fax: +82-43-271-5921;
1578Siddikee et al.
activities such as irrigation . Nearly 20% of the world’s
cultivated land and nearly half of all irrigated lands are
affected by salinity . Salt stress has previously been
reported to cause an increased production of ethylene in
plants, thereby accelerating leaf and petal abscission and
organ senescence, leading to premature death [8, 32, 45].
Reducing the stress-induced ethylene level can alleviate
some of the effects of stresses on plants . In fact, a high
plant loss, of approximatly 40% of photosynthates, is
through root exudates , and it has been postulated that
much of the ACC, which is a precursor of ethylene
produced under stress conditions, may be exuded from
plant roots  and then hydrolyzed by the enzyme ACC
deaminase into ammonia and α-ketobutyrate. This means
that more ACC is exuded by the plant root and drawn
away from the ethylene synthesis pathway , and that
quantities of ACC become lower as ACC oxidase is
converted into ethylene. Thus, PGPR, with ACC deaminase
activity, can be used to reduce the negative effects of
salinity stress [8, 32, 45]. PGPR efficiency is determined
by various environmental factors such as the climate,
weather conditions, soil characteristics, and interaction
with other indigenous microbial flora in the soil .
Mayak et al.  reported that ACC deaminase-producing
salt-tolerant bacteria can survive well in a saline
environment and that their beneficial properties help plants
to overcome stress effects. Halotolerant bacteria are a
group of microorganisms able to grow in media containing
a wide range of NaCl (1-33%) or in the absence of NaCl
. Hence, it was hypothesized that the use of ACC
deaminase-producing PGP halotolerant bacteria could
ameliorate the saline stress effect on plants by reducing
The present study was therefore conducted in an attempt
to isolate and characterize the diverse group of halotolerant
bacteria from coastal soil for their numerous PGP traits.
Selected strains were then checked for their ability to
ameliorate saline stress under gnotobiotic conditions using
MATERIALS AND METHODS
Soil Sampling and Isolation of Halotolerant Bacteria
The sampling sites were situated within 15 km2 of the saline coastal
region of the Yellow Sea near the city of Incheon in the Republic of
Korea. A total of seven soil samples were randomly collected from
either barren fields or the rhizosphere of six different naturally
growing halophytic plants, during the later period of the winter of
2009 (designated as sites 1, 2, and 3 nearest to the coastline; site 4
and sites 5, 6, and 7 about 500 m and 1.5-2 km away from the
coastline, respectively). Three samples were collected from each
site, mixed together to make one composite sample for that site,
sieved at 2 mm in order to separate plant debris and visible fauna,
and then stored at 4oC.
Ten-fold serial dilutions of the samples were made by mixing the
soil with sterile saline water (0.85% NaCl), shaking for 15 min at
150 rpm, and then plating on a tryptic soy agar medium (peptone,
15g/l; tryptone, 5g/l; dextrose, 2.5g/l) modified with 1.75M (~10%)
NaCl, and adjusted to a pH of 8.5 . The plates were incubated at
28oC for 3-4 days, and strains were selected based on colony
morphology, pigmentation, and growth rate. Pure cultures of the
halotolerant bacterial strains were maintained in 30% glycerol at -80oC.
Functional and Biochemical Characterization of Halotolerant
The assay media for the functional and biochemical characterization
of the isolated halotolerant bacterial strains were modified by the
addition of 0.85 M (~5%) NaCl and adjusted to a pH of 7.2.
Nitrogen fixing and sulfur-oxidizing potentials were tested by the
methods described by Gothwal et al.  and Anandham et al. ,
respectively. The indole-3-acetic acid (IAA) and ammonia producing
abilities of the halotolerant bacterial strains were tested by the
method reported by Brick et al.  and Wani et al. , respectively.
The phosphate (P) and zinc (Zn) solubilizing abilities of the
halophiles were tested on Pikovskaya’s medium , which was
supplemented with either 0.5% Ca3(PO4)2 or 0.12% ZnO. Characterization
for extracellular hydrolytic enzyme production was carried out using
recommended media, modified by the addition of 0.85 M NaCl, and
supplemented with the specific substrate for each enzyme: 1.5% (v/v)
colloidal chitin for chitinase, methyl cellulose for cellulase, pectin
for pectinase, tributyrin for lipase, casein for protease, and gelatine
for gelatinase [3, 11]. Urease production was tested using Difco urea
broth (Becton Dickinson Inc., U.S.A.).
Characterization Based on 16S rRNA Gene Sequencing and
For molecular characterization, the selected halotolerant bacterial
strains were subjected to 16S rRNA gene sequence analyses. The
selected bacterial strains were grown in TSA and the DNA was
extracted . The 16S rRNA genes were amplified by a PCR
using the forward primer 27F 5'-AGAGTTTGATCCTGGCTCAG-3'
and the reverse primer 1492R 5'-GGTTACCTTGTTACGACTT-3'.
The 16S rRNA gene sequences were identified by PCR-direct
sequencing, using the fluorescent dye terminator method with ABI
prism equipment and a Bigdye Terminator cycle sequencing ready
reaction kit V.3.1 (Applied Biosystems Inc., U.S.A.), and the
products were purified with a Millipore-Montage dye removal kit
(Millipore-Montage Inc., U.S.A.). Finally, the products were run in
an ABI 3730XL capillary DNA sequencer (Applied Biosystems
Inc., U.S.A.), with a 50 cm capillary.
The obtained 16S rRNA gene sequences were aligned and the
affiliations deduced, using BLAST analysis. Phylogenetic analyses
were performed using MEGA version 4.1  after multiple alignments
of the data by CLUSTAL W . Distances were obtained using
options according to the Kimura two-parameter model , and
clustering was performed using the neighbor-joining method .
The statistical confidence of the nodes was estimated by bootstrapping
using 1,000 replications .
Qualitative Assay of Utilization of ACC
The availability of 1-aminocyclopropane-1-carboxylic acid (ACC) as
a nitrogen source is as a consequence of the enzymatic activity of
ACC deaminase (E.C. 188.8.131.52). ACC deaminase activity was checked
ACC DEAMINASE-PRODUCING HALOTOLERANT BACTERIA AND PLANT GROWTH PROMOTION
according to the method of Glick , with modifications. The
bacteria were first cultured in a TSA medium with 0.85 M NaCl. A
solution of ACC (0.5 M) (Sigma Chemical Co., U.S.A.) was filter-
sterilized (0.2 µm) and frozen at -20oC . Halotolerant bacterial
strains were streaked onto NFb medium supplemented with 3.0 mM
ACC as a nitrogen source. Plates were incubated at 30oC for 4 days.
The ability of a strain to utilize ACC was verified by maintaining
the same strain in a control in the absence of any nitrogen source.
Quantification of ACC Deaminase Activity
ACC deaminase activity was assayed according to the method of
Penrose and Glick , which measures the amount of α-ketobutyrate
produced when the enzyme ACC deaminase cleaves ACC. The
µmole quantity of α-ketobutyrate (Sigma-Aldrich Co., U.S.A.)
produced by this reaction was determined by comparing the
absorbance of a sample to a standard curve of α-ketobutyrate ranging
between 0.1 and 1.0 nmol at 540 nm. A stock solution of α-
ketobutyrate was prepared in 0.1 M Tris-HCl (pH 8.5) and stored at
4oC. In order to measure the specific activity of the cultures, protein
estimation was carried out according to Lowry et al. .
Evaluation of Halotolerant Bacteria Inoculation Effects on
Canola Growth Under Salt Stress in Gnotobiotic Conditions
The halotolerant bacterial strains Br. epidermidis RS15, Br. iodinum
RS16, P. rifietoensis RS18, E. acetylicum RS19, Z. alba RS111, M.
yunnanensis RS222, O. smirnovii RS231, B. stratosphericus ES445,
Br. iodinum RS451, B. stratosphericus RS616, B. licheniformis
RS656, C. variabile RS665, B. aryabhattai ES341, and A. nicotianae
RSA68 were selected for the testing of root elongation and growth
promotion under salt stress in gnotobiotic conditions, on the basis of
the presence or absence of ACC deaminase activity and IAA
production. According to Cheng et al. , the growth of canola
seedlings was reduced by approximately 50% with 150 mM NaCl at
20oC. Hence, this concentration was used to check the bacterial
inoculation effects on the growth promotion of canola. Seed
treatment and gnotobiotic growth pouch assays were performed in
accordance with Penrose and Glick , with slight modifications.
Briefly, halotolerant bacterial strains were grown in TSA
supplemented with 0.85 M NaCl. The cells were then collected and
resuspended in a N-free medium containing 0.85 M NaCl and
supplemented with 5 mM ACC as the sole nitrogen source, and
subsequently incubated for 24 h at 30oC with shaking (120 rpm) in
order to induce ACC deaminase activity. After that, the cells were
harvested, and washed by resuspension in sterile 0.03 M MgSO4.
Canola seeds, Brassica campestris, (Hungnong Seed Co. Ltd.,
Korea and Seminis Korea Inc., Korea), were surface sterilized by
immersion in 70% ethanol for 1 min and 2% NaCl for 30 s,
followed by a thorough rinsing in sterile distilled water (6-7 times).
Then these surface-sterilized seeds were soaked in sterile distilled
water, or bacterial suspension (1×108CFU/ml), for 24 h. Following
on from this, 20 ml of water, or water containing 150 mM NaCl,
was added to CYG seed germination pouches (Mega International
Manufacturer Inc., U.S.A.) and autoclaved at 121oC for 15 min.
Sprouted seeds were transferred aseptically to growth pouches and
incubated in a DS 54 GLP growth chamber (DASOL Scientific Co.,
Ltd., Korea) and maintained at 20±1oC, with a relative humidity of
70% and a dark/light cycle beginning with 12 h of dark, followed
by 12 h of light (18 µmol m-2 s-1). Seven seeds were placed in each
pouch for each treatment, and each treatment had three replicates.
Seeds in growth pouches treated only with water were used as the
negative control, and seeds in growth pouches treated only with salt
solution were used as the positive control. In theory, the positive
controls should be found to reduce growth in a manner very similar
to the actual salt stress effects on plants in the field, whereas
negative controls should provide the standard for any changes from
the normal growth process of the plant. Data were collected
regarding root length and the total dry weight of the plants from 7-
day-old canola seedlings.
Data on the growth parameters of canola were subjected to analysis
of variance (ANOVA). Significance at the 5% level was tested by
Duncan’s multiple range test (DMRT) using the SAS package,
Version 9.1.3 (SAS, 2010).
Nucleotide Sequence Accession Numbers
The National Center for Biotechnology Information GenBank
accession numbers for the sequences of halotolerant bacterial strains
are from GU968455 through to GU968490.
Functional and Biochemical Characterization of
One hundred and forty halotolerant bacterial stains, isolated
from seven soil samples collected from coastal soil, were
able to grow on medium containing 1.75 M (10%) NaCl.
Of these, Table 1 illustrates the 36 strains that possessed
multiple PGP traits, and were selected for further study.
16S rRNA Gene Sequencing and Phylogenetic
The selected halotolerant bacterial strains were identified
by 16S rRNA gene sequencing analysis to ascertain their
taxonomic positions. The nucleotide sequences recovered
from these bacterial strains were subjected to homology in
the NCBI database, which showed that the representative
36 halotolerant bacterial strains belong to 3 phyla, 4
orders, 7 families, and 10 different genera (Fig. 1). Out of
the 36 strains, 12 strains (RS652, RS616, RS654, ES445,
ES446, RS233, RS340, RS447, RS114, RS656, RS341,
and ES667) showed a 99-100% similarity with the 16S
rRNA gene sequences of the genus Bacillus, 8 strains
(RS263, RS212, RS449, RS451, RS448, RS16, RS15, and
RS361) showed a 98-99.2% similarity to Brevibacterium,
5 strains (RS18, RS224, RS344, RS338, and RS112)
exhibited likeness at ≤99% to Planococcus, 3 strains
(RS234, RS236, and RS111) at ≤98% to Zhihengliuella, 2
strains (RS343 and RS19) at ≤99% to Exiguobacterium, 2
strains (ES11E and RS229) at 96-99% to Halomonas, 1
strain (RS231) at ≤98% to Oceanimonas, 1 strain (ES665)
at ≤98% to Corynebacterium, 1 strain (RSA68) at ≤99% to
Arthrobacter, and 1 strain (RS222) at ≤99% to Micrococcus
(Table 1). The first phylum under Firmicutes had a close
Siddikee et al.
Table 1. Identification and characteristics of halotolerant bacterial strains isolated from coastal soil.
Plant growth promoting traitsb
Protease Pectinase ChitinaseCellulaseLipase
RS15 Brevibacterium epidermidis (99.1)GU968456 2.37±0.48 +++++--++
RS16 Brevibacterium iodinum (99.0)GU968457 4.13±1.05 +++++----
RS18Planococcus rifietoensis (99.8)GU968458 -+-++---+-
RS19Exiguobacterium acetylicum (99.5)GU968459 -+-++---++
RS111 Zhihengliuella alba (98.3)GU9684601.38±0.86 ++++-++--
RS112Planococcus rifietoensis (99.7)GU968461 -++++--+++
RS114Bacillus pumilus (99.7)GU968462 -+-+++----
ES11EHalomonas neptunia (99.7)
RS222Micrococcus yunnanensis (99.7) GU968463 0.82±0.09++++-+-+-
RS212Brevibacterium epidermidis (99.1) GU968464 2.25±0.51 ++++-++--
RS224Planococcus rifietoensis (99.9)GU968465 0.96±0.06 +-++--+--
RS229Halomonas korlensis (96.2)GU968466 -++-+-----
RS231Oceanimonas smirnovii (98.9)GU9684671.40±0.51 ++++----+
RS233Bacillus stratosphericus (100)GU9684681.93±0.54 ++++--+++
RS234Zhihengliuella alba (98.3)GU968469 -++++-----
RS236Zhihengliuella alba (98.3)GU9684700.73±0.06 ++++--+--
RS263 Brevibacterium epidermidis (99.2) GU968486 -++++-+-+-
RS338Planococcus rifietoensis (99.9)GU968471 -+++++-+--
RS340 Bacillus stratosphericus (100)GU968472 -+-++++---
RS341Bacillus aryabhattai (99.9) GU9684731.11±0.42 +-++-+---
RS343 Exiguobacterium acetylicum (99.5)GU968474 0.99±0.09 ++++--+-+
RS344 Planococcus rifietoensis (99.9)GU968475 -+-++-+---
RS361Brevibacterium epidermidis (98.9)GU9684851.27±0.81++++-++--
ES445Bacillus stratosphericus (100) GU9684761.19±0.18+-++--++-
ES446Bacillus stratosphericus (100)GU9684772.89±0.87+-+++--+-
RS447Bacillus pumilus (99.7)GU9684780.89±0.06+-+++-+--
RS448Brevibacterium epidermidis (99)GU9684794.90±1.0 ++++-----
RS449Brevibacterium epidermidis (99.1)GU9684800.69±0.27 ++++-----
RS451Brevibacterium iodinum (98.6)GU9684811.11±0.12 ++++-----
RS652Bacillus stratosphericus (99.7)GU9684822.01±0.63 +++++----
RS616Bacillus stratosphericus (99.7) GU968483 -++++-----
RS654 Bacillus stratosphericus (99.9)GU9684841.63±0.21 +++++--++
RS656 Bacillus licheniformis (99.7)GU9684903.06±1.14 +-++-----
RS665Corynebacterium variabile (98.3)GU9684871.17±0.45 +-+++----
ES667Bacillus aryabhattai (100)GU9684881.37±0.15 +-++++---
RSA68Arthrobacter nicotianae (99.2) GU9684890.70±0.06 +++++----
aACC deaminase activity: µmol α-ketobutyrate mg protein-1 h-1; values are means ± SD of three replications.
bPlant growth promoting traits: NF=Nitrogen fixation; IAA=Indole acetic acid production; S2O3=Thiosulfate oxidation; NH3=Ammonia production; + (plus) indicates presence; - (minus) indicates
absence of respective traits tested.
ACC DEAMINASE-PRODUCING HALOTOLERANT BACTERIA AND PLANT GROWTH PROMOTION
Fig. 1. Phylogenetic tree based on a 16S rRNA gene sequence analysis.
The halotolerant bacterial sequence is shown in bold font. The scale bar represents the expected number of substitutions.
sequence similarity, with a low DNA G+C content, to genera
Bacillus, Planococcus, and Exiguobacterium; the second
phyla under Actinobacteria, with a high DNA G+C content,
was composed of the genera Brevibacterium, Zhihengliuella,
Arthrobacter, Micrococcus, and Corynebacterium; and the
third phyla Proteobacteria comprised the genera Halomonas
and Oceanimonas (Fig. 1). Strain RS229 showed a 96%
sequence homology with Halomonas korlensis in the
database, indicating that it may belong to a new species of
1582 Siddikee et al.
ACC Deaminase Activity
The 36 strains possessing multiple PGP traits were tested
for ACC deaminase activity, and 25 strains were found to
be positive. Quantification of ACC deaminase activity
showed wide variations (Table 1). The activity was highest
in the cell-free extracts obtained from Br. epidermidis
RS448 (4.9 µmol α-ketobutyrate mg protein-1 h-1) followed
by Br. iodinum RS16 (4.1 µmol α-ketobutyrate mg protein-1
h-1), B. licheniformis RS656 (3 µmol α-ketobutyrate mg
protein-1 h-1), B. stratosphericus RS446 (2.9 µmol α-
ketobutyrate mg protein-1 h-1), Br. epidermidis RS15
(2.4µmol α-ketobutyrate mg protein-1 h-1), and Br. epidermidis
RS212 (2.3 µmol α-ketobutyrate mg protein-1 h-1) (Table 1).
Halotolerant Bacterial Inoculation Effects on Canola
Growth Under Salt Stress in Gnotobiotic Conditions
Most of the tested strains were able to significantly
promote the growth of canola seedlings in the presence of
salt. Salt stress (150 mM NaCl) reduced root length by
37% and the plant total dry weight biomass by 40% in 7-
day-old canola seedlings when compared with the negative
control. Other than B. stratosphericus RS616, P. rifietoensis
RS18, and E. acetylicum RS19, all of the 14 tested
halotolerant bacterial strains were able to improve root
length by between 29% and 47%, and dry weight by
between 35% and 43% when under salt-stressed conditions
in 7-day-old canola seedlings when compared with the
positive control (Table 2).
PGPR stimulate plant growth via both direct and indirect
mechanisms with variable results depending on a number
of environmental factors [12, 15, 16]. In the current study,
a large number of halotolerant bacteria were characterized
as being positive for nitrogen fixation, IAA production, P
and Zn solubilization, S oxidation, and NH3 production.
Upadhyay et al.  found that only 18% (24 out of 130)
of strains tested could tolerate up to 8% of NaCl, while
maintaining the ability to produce IAA, fix nitrogen, and
solubilize P. It is possible that extracellular hydrolytic
enzymes may indirectly promote plant growth [16, 40],
and Rohban et al.  reported that a large number of
halotolerant bacteria were found positive for extracellular
hydrolytic enzyme production. These findings agree with
other studies [5, 42], where a number of halotolerant
bacteria were found to have multiple PGP traits.
Phylogenetic analysis of the halotolerant bacterial 16S
rRNA gene sequence revealed that highly diverse bacterial
populations exist in coastal soils, representing some 10
different genera (Table 1 and Fig. 1). From 16S rRNA
analysis, one third of the halotolerant bacteria exhibited 98
-100% sequence homology with known 16S rRNA of the
cultivated Bacillus genus (Table 1 and Fig. 1). In the
course of the characterization of microorganisms present
in a tidal flat of the Yellow Sea in Korea, many moderately
halotolerant/halophilic bacteria have been isolated and
characterized taxonomically as belonging to Bacillus .
PGPR that have ACC deaminase activity help plants to
withstand stress (biotic or abiotic) by reducing the level
of stress ethylene [8, 32, 45]. In the present study, we
screened halotolerant bacteria having ACC deaminase
activity with multiple PGP traits, and found that 25 out of
the screened 36 strains showed ACC deaminase activity.
Variations in levels of ACC deaminase activity of the strains
were noted, and these results are concurrent with earlier
findings [5, 21]. All of the genera (except Planococcus)
isolated exhibited ACC deaminase activity. Thus, this is
consistent with previous observations that diverse groups
of bacteria were found to exhibit ACC deaminase activity
[5, 21, 34].
Several reports show that ACC deaminase-producing
PGPR increase root elongation and plant growth by
reducing the ethylene stress [8, 13-15, 32, 45]. In this study,
14 halotolerant bacterial strains were tested for their
growth promoting activity, under axenic conditions at
150 mM NaCl, by conducting growth pouch experiments
on canola. Halotolerant bacteria, having the ability to
produce both ACC deaminase and IAA, were found to
enhance root elongation and the dry weight of canola to a
greater extent than strains that produced solely ACC
deaminase. It is likely that auxin and ACC deaminase
Table 2. Halotolerant bacterial inoculation effects on canola
growth under salt stress in gnotobiotic condition.
Brevibacterium epidermidis RS15
Brevibacterium iodinum RS16
Planococcus rifietoensis RS18
Exiguobacterium acetylicum RS19
Arthrobacter nicotianae RSA68
Zhihengliuella alba RS1111.03±0.03abc
Micrococcus yunnanensis RS222
Oceanimonas smirnovii RS231
Bacillus licheniformis RS656
Bacillus stratosphericus ES445
Bacillus aryabhattai RS341
Bacillus stratosphericus RS616
Corynebacterium variabile RS665
Bacillus aryabhattai ES667
*Values (mean±SD *r=3) with the same letters are not significantly
different at p≤0.05.
ACC DEAMINASE-PRODUCING HALOTOLERANT BACTERIA AND PLANT GROWTH PROMOTION
stimulate root growth in a coordinated fashion [13, 28].
Since IAA secreted by a bacterium may promote root
growth directly, by stimulating plant cell elongation or cell
division , this observation is consistent with the model
 that envisions a complex cross-talk between IAA and
ethylene in the promotion of plant growth by PGPR. The
fact that halotolerant bacterial strains, which produce IAA
but not ACC deaminase, inhibit root growth rather than
promote elongation in the presence of salt might reflect a
higher synthesis rate of ACC under stress. Moreover, IAA
stimulates ACC synthase transcription, which, in the
absence of ACC deaminase, could result in enhanced
ethylene biosynthesis. In concordance with the results of
the present study, Cheng et al.  showed that a wild-type
strain of Pseudomonas putida UW4 (which produces IAA
and ACC deaminase), but not an ACC deaminase minus
mutant of this bacterium, protected canola seedlings from
growth inhibition by high levels of salt.
In conclusion, our work has demonstrated that different
halotolerant bacteria, isolated from soils obtained in a
barren field and from the rhizosphere of halophytes, are
able to withstand high salt concentrations (1.75 M NaCl)
and pH (6.5 to 8.5), and can facilitate plant growth
promotion in the presence of growth inhibitory levels of
salt. These results further suggest, given the variation in
activity observed, that the selection and subsequent
industrial use of ACC deaminase-producing halotolerant
bacteria, with multiple PGP activities for the facilitation
of plant growth in saline environments, will be a highly
important area for future research. Hence, further evaluation
of these halotolerant bacterial strains is needed to uncover
their efficiency as plant growth promoting bacteria in soil-
This work was supported by the Mid-Career Researcher
Program through an NRF grant funded by the MEST (No.
2010-0000418). Md. Ashaduzzaman Siddikee would like
to convey a special thanks to Brain Korea21 (BK21) for
their award of a Ph.D. fellowship. All of the authors are
also very grateful to Prof. Bernard R. Glick of the
University of Waterloo in Canada, and Dr. M. Madhaiyan
of the Temasek Life Sciences Laboratory at the National
University of Singapore, for their critical review and
helpful suggestions for this manuscript.
1. Abrol, I. P., J. S. P. Yadav, and F. I. Massoud. 1988. Salt
Affected Soils and Their Management, p. 39. Food and
Agriculture Organization (FAO), UN, Soils Bulletin, Rome.
2. Anandham, R., R. Sridar, P. Nalayini, S. Poonguzhali, M.
Madhaiyan, and T. M. Sa. 2007. Potential for plant growth
promotion in groundnut (Arachis hypogaea L.) cv. ALR-2 by
co-inoculation of sulfur-oxidizing bacteria and Rhizobium.
Microbiol. Res. 162: 139-153
3. Basha, S. and K. Ulaganathan. 2002. Antagonism of Bacillus
species (strains 121) towards Curvularia lunata. Curr. Sci. 82:
4. Bayliss, C., E. Bent, D. E. Culham, S. MacLellan, A. J. Clarke,
G. L. Brown, and J. M. Wood. 1997. Bacterial genetic loci
implicated in the Pseudomonas putida GR12-2R3-canola
mutualism: Identification of an exudate-inducible sugar transporter.
Can. J. Microbiol. 43: 809-818.
5. Belimov, A. A., V. I. Safronova, T. A. Sergeyeva, T. N. Egorova,
V. A. Matveyeva, V. E. Tsyganov, et al. 2001. Characterization
of plant growth promoting rhizobacteria isolated from polluted
soils and containing 1-aminocyclopropane-1-carboxylate
deaminase. Can. J. Microbiol. 47: 642-652.
6. Brick, J. M., R. M. Bostock, and S. E. Silverstone. 1991. Rapid
in situ assay for indole acetic acid production by bacteria
immobilized on nitrocellulose membrane. Appl. Environ. Microbiol.
7. Brisou, J., D. Courtois, and F. Denis. 1974. Microbiological study
of a hypersaline lake in French Somaliland. Appl. Microbiol.
8. Cheng, Z., E. Park, and B. R. Glick. 2007. 1-Aminocyclopropane-
1-carboxylate deaminase from Pseudomonas putida UW4
facilitates the growth of canola in the presence of salt. Can. J.
Microbiol. 53: 912-918.
9. DasGupta, S. M., N. Khan, and C. S. Nautiyal. 2006. Biologic
control ability of plant growth-promoting Paenibacillus lentimorbus
NRRL B-30488 isolated from milk. Curr. Microbiol. 53: 502-505.
10. Felsenstein, J. 1985. Confidence limits on phylogenies: An
approach using the bootstrap. Evolution 39: 783-791.
11. Gerhardt, P., R. G. E. Murray, W. A. Wood, and N. R. Krieg.
1994. In: Methods for General and Molecular Bacteriology.
American Society for Microbiology, Washington, DC.
12. Giongo, A., A. Ambrosini, L. K. Vargas, J. R. J. Freire, M. H.
Bodanese-Zanettini, and L. M. P. Passaglia. 2008. Evaluation of
genetic diversity of Bradyrhizobia strains nodulating soybean
[Glycine max (L.) Merrill] isolated from South Brazilian fields.
Appl. Soil Ecol. 38: 261-269.
13. Glick, B. R., Z. Cheng, J. Czarny, and J. Duan. 2007.
Promotion of plant growth by ACC deaminase-producing soil
bacteria. Eur. J. Plant Pathol. 119: 329-339
14. Glick, B. R. 2004. Bacterial ACC deaminase and the alleviation
of plant stress. Adv. Appl. Microbiol. 56: 291-312.
15. Glick, B. R., D. M. Penrose, and J. Li. 1998. A model for the
lowering of plant ethylene concentrations by plant growth
promoting bacteria. J. Theor. Biol. 190: 63-68.
16. Glick, B. R. 1995. The enhancement of plant growth by free-
living bacteria. Can. J. Microbiol. 41: 109-117.
17. Grichko, V. P. and B. R. Glick. 2001. Amelioration of flooding
stress by ACC deaminase-containing plant growth-promoting
bacteria. Plant Physiol. Biochem. 39: 11-17.
18. Gothwal, R. K., V. K. Nigam, M. K. Mohan, D. Sasmal, and P.
Ghosh. 2007. Screening of nitrogen fixers from rhizospheric
bacterial strains associated with important desert plants. Appl.
Ecol. Environ. Res. 6: 101-109.
1584 Siddikee et al.
19. Hariprasad, P. and S. R. Niranjana. 2009. Isolation and
characterization of phosphate solubilizing rhizobacteria to improve
plant health of tomato. Plant Soil 316: 13-24.
20. Indiragandhi, P., R. Anandham, K. Kim, W. Yim, M. Madhaiyan,
and T. M. Sa. 2008. Induction of defense responses in tomato
against Pseudomonas syringae pv. tomato by regulating the
stress ethylene level with Methylobacterium oryzae CBMB20
containing 1-aminocyclopropane-1-carboxylate deaminase. World
J. Microbiol. Biotechnol. 24: 1037-1045.
21. Indiragandhi, P., R. Anandham, M. Madhaiyan, and T. M. Sa.
2007. Characterization of plant growth-promoting traits of
bacteria isolated from larval guts of diamond back moth
Plutella xylostella (Lepidoptera: Plutellidae). Curr. Microbiol.
22. Iqbal, U., N. Jamil, I. Ali, and S. Hasnain. 2010. Effect of zinc-
phosphate-solubilizing bacterial strains on growth of Vigna
radiata. Ann. Microbiol. 61: 1869-2044.
23. Kang, S. M., G. J. Joo, M. Hamayun, C. I. Na, D. H. Shin, H.
Y. Kim, J. K. Hong, and I. J. Lee. 2009. Gibberellin production
and phosphate solubilization by newly isolated strains of
Acinetobacter calcoaceticus and its effect on plant growth.
Biotechnol. Lett. 31: 277-281.
24. Kimura, M. 1980. A simple method for estimating evolutionary
rates of base substitutions through comparative studies of
nucleotide sequences. J. Mol. Evol. 16: 111-120.
25. Krause, M. S., T. J. J. De-Ceuster, S. M. Tiquia, F. C. Jr.
Michel, L. V. Madden, and H. A. J. Hoitink. 2003. Isolation and
characterization of rhizobacteria from composts that suppress
the severity of bacterial leaf spot of radish. Phytopathology 93:
26. Kumar, S., K. Tamura, and M. Nei. 2004. MEGA3: Integrated
software for molecular evolutionary genetics analysis and
sequence alignment. Brief Bioinform. 5: 150-163.
27. Larsen, H. 1986. Halophilic and halotolerant microorganisms-
an overview and historical perspective. FEMS Microbiol. Rev.
28. Li, J., D. H. Ovakim, T. C. Charles, and B. R. Glick. 2000. An
ACC deaminase minus mutant of Enterobacter cloacae UW4
no longer promotes root elongation. Curr. Microbiol. 41: 101-105.
29. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.
1951. Protein measurement with Folin-phenol reagent. J. Biol.
Chem. 193: 265-275.
30. Lynch, J. M. and J. M. Whipps. 1991. Substrate flow in the
rhizosphere, pp. 15-24. In D. L. Keister and B. Cregan (eds.).
The Rhizosphere and Plant Growth. Kluwer, Dordrecht.
31. Madhaiyan, M., S. Poonguzhali, J. Ryu, and T. M. Sa. 2006.
Regulation of ethylene levels in canola (Brassica campestris)
by 1-aminocyclopropane-1-carboxylate deaminase-containing
Methylobacterium fujisawaense. Planta 224: 268-278.
32. Mayak, S., T. Tirosh, and B. R. Glick. 2004. Plant growth-
promoting bacteria confer resistance in tomato plants to salt
stress. Plant Physiol. Biochem. 42: 565-572.
33. Patten, C. L. and B. R. Glick. 2002. Role of Pseudomonas
putida indoleacetic acid in development of the host plant root
system. Appl. Environ. Microbiol. 68: 3795-3801.
34. Penrose, D. M. and B. R. Glick. 2003. Methods for isolating
and characterizing ACC deaminase-containing plant growth-
promoting rhizobacteria. Physiol. Plant 118: 10-15.
35. Pikovskaya, R. I. 1948. Mobilization of phosphorus in soil in
connection with the vital activity of some microbial species.
Mikrobiologiya 17: 362-370.
36. Reed, M. L. E. and B. R. Glick. 2005. Growth of canola
(Brassica napus) in the presence of plant growth-promoting
bacteria and either copper or polycyclic aromatic hydrocarbons.
Can. J. Microbiol. 51: 1061-1069.
37. Rohban, R., M. A. Amoozegar, and A. Ventosa. 2009.
Screening and isolation of halotolerant bacteria producing
extracellular hydrolyses from Howz Soltan Lake, Iran. J. Ind.
Microbiol. Biotechnol. 36: 333-340.
38. Sambrook, J., E. F. Fritsch, and T. Maniatis, (eds.). 1989.
Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring
Harbor Laboratory Press, New York, USA.
39. Saitou, N. and M. Nei. 1987. The neighbor-joining method: A
new method for reconstructing phylogenetic trees. Mol. Biol.
Evol. 4: 406-425.
40. Swain, M. R., R. C. Ray, and C. S. Nautiyal. 2008. Biocontrol
efficacy of Bacillus subtilis strains isolated from cow dung
against postharvest yam (Dioscorea rotundata L.) pathogens.
Curr. Microbiol. 57: 407-411.
41. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994.
CLUSTAL W: Improving the sensitivity of progressive multiple
sequence alignment through sequence weighting, position specific
gap penalties and weight matrix choice. Nucleic Acids Res. 22:
42. Upadhyay, S. K., D. P. Singh, and R. Saikia. 2009. Genetic
diversity of plant growth promoting rhizobacteria isolated from
rhizospheric soil of wheat under saline condition. Curr. Microbiol.
43. Wani, P. A., M. S. Khan, and A. Zaidi. 2007. Chromium
reduction, plant growth-promoting potentials, and metal
solubilization by Bacillus sp. isolated from alluvial soil. Curr.
Microbiol. 54: 237-243.
44. Waino, M., B. J. Tindall, P. Schumann, and K. Ingvorsen. 1999.
Gracilibacillus gen. nov., with description of Gracilibacillus
halotolerans gen. nov., sp. nov.; transfer of Bacillus dipsosauri
to Gracilibacillus dipsosauri comb. nov., and Bacillus salexigens
to the genus Salibacillus gen. nov., as Salibacillus salexigens
comb. nov. Int. J. Syst. Bacteriol. 49: 821-831.
45. Zahir, A. Z., U. Ghani, M. Naveed, S. M. Nadeem, and H. N.
Asghar. 2009. Comparative effectiveness of Pseudomonas and
Serratia sp. containing ACC-deaminase for improving growth
and yield of wheat (Triticum aestivum L.) under salt-stressed
conditions. Arch. Microbiol. 191: 415-424.
46. Zhu, J. K. 2001. Plant salt tolerance. Trends Plant Sci. 6: 66-71.