Antonie van Leeuwenhoek

DOI 10.1007/s10482-015-0445-z

ORIGINAL PAPER

Isolation and characterization of endophytic plant growthpromoting bacteria from date palm tree (Phoenix dactylifera
L.) and their potential role in salinity tolerance
Mahmoud W. Yaish Irin Antony
Bernard R. Glick

Received: 29 September 2014 / Accepted: 1 April 2015

Springer International Publishing Switzerland 2015

Abstract Endophytic bacteria were isolated from

date palm (Phoenix dactylifera L.) seedling roots,
characterized and tested for their ability to help plants
grow under saline conditions. Molecular characterization showed that the majority of these strains belonged
to the genera Bacillus and Enterobacter and had
different degrees of resistance to various antibiotics.
Some of these strains were able to produce the enzyme
1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and the plant growth regulatory hormone
indole-3-acetic acid (IAA). Some strains were also
able to chelate ferric iron (Fe3?) and solubilize
2?
potassium (K?), phosphorus (PO34 ) and zinc (Zn ),
and produce ammonia. The results also showed that
ACC deaminase activity and IAA production was
slightly increased in some strains in response to an
increase in NaCl concentration in the growth media.
Consistent with these results, selected strains such as
PD-R6 (Paenibacillus xylanexedens) and PD-P6

Electronic supplementary material The online version of

this article (doi:10.1007/s10482-015-0445-z) contains supplementary material, which is available to authorized users.
M. W. Yaish (&)
I. Antony
Department of Biology, College of Science, Sultan
Qaboos University, Muscat, Oman
e-mail: myaish@squ.edu.om
B. R. Glick
Department of Biology, University of Waterloo,
Waterloo, ON N2L 3G1, Canada

(Enterobacter cloacae) were able to enhance canola

root elongation when grown under normal and saline
conditions as demonstrated by a gnotobiotic root
elongation assay. These results suggest that the
isolated and characterized endophytic bacteria can
alter ethylene and IAA levels and also facilitate
nutrient uptake in roots and therefore have the
potential role to promote the growth and development
of date palm trees growing under salinity stress.
Keywords Plantmicrobe interactions

Endophytes
Date Palm
1-aminocyclopropane-1carboxylic acid
Salinity

Introduction
Endophytic microbial communities play a significant
role in the growth and development of host plants
growing under both normal and stress conditions.
Endophytic bacteria have the ability to colonize
internal plant tissues without causing any disease or
damage. Indeed, these bacteria may improve plant
growth and developmental processes (Glick 1995;
Glick et al. 1998; Ryan et al. 2008; Schulz and Boyle
2006) by producing a range of nutrient products and
facilitating primary and secondary nutrient uptake
through atmospheric nitrogen fixation (Gothwal et al.
2008), the formation of iron siderophores (Wang et al.
1993) and the solubilization of minerals such as

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Antonie van Leeuwenhoek

phosphate (PO34 ) (Hariprasad and Niranjana 2009;

Kang et al. 2009), potassium (K?) (Basak and Biswas
2009; Sugumaran and Janarthanam 2007) and zinc
(Zn2?) (Iqbal et al. 2010) in the rhizosphere. Endophytes may also supply roots with growth-promoting
phytohormones such as auxin (Rashid et al. 2012;
Siddikee et al. 2010), cytokinin (Bhore et al. 2010;
Lichter et al. 1995; Powell and Morris 1986) and
gibberellin (Kang et al. 2009). Furthermore, some
bacteria inhibit the production of the plant hormone
ethylene, which is usually generated in response to a
wide variety of environmental stresses, including
pathogen infections (Indiragandhi et al. 2008; Wang
et al. 2000), drought (Mayak et al. 2004b; Zahir et al.
2008) and soil salinity (Cheng et al. 2007; Mayak et al.
2004a; Siddikee et al. 2010; Wang et al. 1993; Zahir
et al. 2009).
Plants growing in saline soil frequently increase
their ethylene production in order to initiate programmed cell death (apoptosis) (Trobacher 2009),
which in turn leads to tissue senescence. Some
endophytic bacteria have the ability to produce the
enzyme 1-aminocyclopropane-1-carboxylic acid
(ACC) deaminase that breaks down ACC, the direct
precursor of ethylene, into ammonia and a-ketobutyrate. In this process, bacteria use ACC as a source of
nitrogen and thereby reduce the deleterious effect of
ethylene on plant tissues (Glick et al. 2007).
Soil salinity alters various physiological processes
in plants, including the imbalance of nutrients taken up
by roots (Alam and Pessarakli 1999; Talei et al. 2012)
and the production of additional ethylene. Typically,
the presence of endophytic bacteria is important for
plant growth and development; however, the bacterias importance is greater when plants are exposed to
environmental stress conditions. Under these conditions, the availability of auxin, ACC deaminase and
the nutrients produced by these bacteria is critical to
minimize the consequences of physiological stress and
to maintain adequate nutrition to support the level of
growth and development required to complete the
lifecycle of the plants (Timmusk et al. 2011).
The date palm (Phoenix dactylifera L.) is a primary
fruit crop in the Middle East and North Africa,
including the Persian Gulf countries. The date palm
tree has long been considered an important source of
food and is consumed as fruit, syrup and livestock
feed. In recent times, the date palm tree has suffered
from the excessive amounts of salts that have

123

accumulated in soil due to anthropogenic activities

such as over-irrigation using underground saline
water. Soil salinity is a global agricultural problem:
about 20 % of cultivated lands and 50 % of irrigated
areas are affected by salinity (Munns and Tester 2008;
Peleg et al. 2011; Zhu 2001). Despite the fact that
some date palm varieties have the ability to adapt to
relatively high salinity levels (up to 12.8 dS m-1)
(Ramoliya and Pandey 2003), excessively high soil
salinity causes a significant loss in the quantity and
quality of yields (Alhammadi and Kurup 2012). The
study reported here was designed to isolate and
characterize endophytic bacteria endemic to date
palms and to identify some of the mechanisms that
these bacteria may use in facilitating date palm growth
in saline environments.

Materials and methods

Isolation of endophytic root bacteria
A total of four different soil samples were collected
from the rhizosphere of date palm trees growing in
orchards located in different geographical locations in
Oman, specifically on the campus of the Sultan
Qaboos University (Muscat), Al-sharqia, Al-Dakhilia
and Al-Batinah regions, Oman. These soils were used
to grow date palm seeds (Khalas variety). After
thoroughly washing them with water, date palm seeds
were surface sterilized with 75 % ethanol for 10 min
and then by a 5.25 % solution of commercial bleach
for 10 min. Subsequently, seeds were thoroughly
rinsed, once with sterile water supplemented with
10 % Tween-20, and then 4 times with sterile distilled
water. Surface sterilized seeds were soaked overnight
in sterile distilled water and then planted in 1 l pots of
soil. Five pots were planted for each soil sample and
incubated in a growth chamber under controlled
environmental condition with 16/8 h day/night, at
35/30 C, respectively. After 6 weeks, seedling roots
growing in each soil sample were separated from the
plants, pooled, surface sterilized (Rashid et al. 2012)
and the endophytic bacteria were eluted from the
tissues using Ringers solution as described (Rashid
et al. 2012). The eluted Ringers solutions were diluted
and 10 ll aliquots were spread onto Kings B agar
(Kg) (20 g proteose peptone 3, 10 ml glycerol, 1.5 g
K2HPO4, 1.5 g MgSO4
7H2O and 15 g agar in 1 l of

Antonie van Leeuwenhoek

water), Luria agar (LA) (Sigma) and tryptic soy agar

(TSA) rich media (Difco Laboratories). To confirm the
absence of any bacteria on the rhizoplane after
disinfection, 24 cm pieces of roots were placed
directly on agar media of different types (Kg, LA and
TSA) and incubated at 32 C for 5 days.
In order to isolate endophytes with ACC
deaminase activity, the previously described method
was used with a few modifications (Penrose and
Glick 2003). Briefly, 1 ml of the eluted Ringers
solution of every four samples prepared from root
tissues of the previous experiment was pooled into
one tube and 1 ml of this was used to inoculate
Pseudomonas Agar F (PAF) medium (per liter of
water: 10 g proteose peptone, 10 g casein hydrolysate, 1.5 g anhydrous MgSO4, 1.5 g K2HPO4
and 10 ml glycerol). After 24 h of incubation at
32 C with shaking, 1 ml aliquot of the culture was
used to inoculate minimal growth medium (MM)
DF salts (Dworkin and Foster 1958) containing
ammonium acetate as a nitrogen source. Subsequently a 1 ml aliquot of the culture was used to
inoculate minimal growth media (MM) containing
ACC as the sole nitrogen source and glucose and
gluconic acid as sources of carbon (Penrose and
Glick 2003). After 48 h of incubation with shaking
at 32 C, aliquots of the liquid culture were
streaked onto MM containing 6 mM ACC and
incubated at 32 C for 72 h. Based on colony
morphology and abundance, one or two colonies
were isolated per petri dish, sub-cultured and used
for the following molecular characterization and
enzymatic assays.
The optimum temperature of these strains was
determined based on the growth curve which was
constructed by plotting the optical density (O.D.) at
several time points within 72 h. All strains have an
optimum growth temperature between 30 and 35 C.
Therefore, 32 C was used as the growth temperature
in all subsequent experiments.
To prove that the isolated strains are true endophytes, canola seeds were inoculated with individual
bacteria isolates and subsequently these strains were
separately re-isolated from seedlings according to the
previously described method (Rashid et al. 2012;
Rosenblueth and Martinez-Romero 2006) where
Pseudomonas putida UW4, a rhizospheric, non-endophytic plant growth-promoting strain was used as a
negative control.

Strain identification based on 16S rDNA gene

sequences
Because all strains were isolated from date palm roots,
they were given the letters PD which stands for the
acronym of the genus and species of this plant
Phoenix dactylifera L.. The R and L letters were
given for those strains isolated using the first strategy,
while P was given for the strains isolated using the
second strategy (see above). The numbers that follow
the letters are serial numbers given to each strain. As
for stains identification, total genomic DNA was
extracted using the boiling phosphate buffer saline
(PBS) method in which separate colonies were
inoculated in tryptic soy broth (TSB) (Difco Laboratories) medium and incubated overnight at 32 C with
rotary shaking at 220 rpm. Then the cells in the culture
medium were harvested by centrifugation at 5000 rpm
for 15 min, washed with 0.5 ml phosphate buffered
saline (PBS) (Sigma) and centrifuged again. Subsequently, the cell pellets were re-suspended with 0.1 ml
PBS and heated up to 99 C for 15 min using a
thermocycler (Applied Biosystems) and were centrifuged again at 5000 rpm for 30 min. The supernatants were used as a source of DNA template in the
PCR to amplify the 16S rRNA gene using 27F and
1492R primers for bacteria (Lane 1991). PCR products
were run on a 1 % agarose gel, excised, purified and
sequenced using 27F and 1492R primers. A 400 bp
fragment of the 16S rRNA gene sequence was used to
identify each strain using the ribosomal DNA database
available at the National Institute of Biotechnology.
ACC deaminase activity and IAA production
The capacity of newly isolated strains to produce ACC
deaminase was measured as described (Penrose and
Glick 2003) using a standard curve of a-ketobutyrate
(Sigma) between 0.1 and 1.0 lM. To study the effect
of NaCl on ACC deaminase activity, NaCl was added
to MM medium supplemented with 6 mM ACC at
final concentrations of 0, 50, 100 and 200 mM NaCl.
A 10 ll aliquot of a bacterial culture (O.D600 = 0.8)
was used to inoculate 1 ml MM medium. The culture
was incubated at 32 C with shaking until it reached
the late-exponential growth stage (O.D600 = 1). IAA
production was determined by the capacity of these
strains to utilize 500 lg ml-1 L-tryptophan in MM to
produce IAA and similar molecules. To test the effect

123

Antonie van Leeuwenhoek

of salinity on IAA production, MM was also supplemented with 0, 50, 100 and 200 mM NaCl. A 10 ll
aliquot of a bacterial culture (O.D600 = 0.8) was used
to inoculate 50 ml MM and the IAA and similar
compounds were measured in the supernatant during
the late exponential stage at (O.D600 = 1) as this stage
was experimentally defined for the tested strains.
The production of IAA was colorimetrically measured for all isolated strains as previously described
(Glickmann and Dessaux 1995) and using Salkowskis
reagent (Gordon and Weber 1951) as the colorimetric
reagent. The strains were also inoculated in MM
medium lacking tryptophan which was used as a
negative control in the experiment. The IAA content
of each sample was estimated using a standard curve
ranging from 0.01 to 0.4 mg ml-1 IAA (Sigma).
Production of ammonia
The capacity of the newly isolated endophytes to
produce ammonia was measured as previously described (Marques et al. 2010) by growing the bacteria
in 1 % proteose peptone at 32 C for 24 h followed by
staining the culture with Nesslers reagent in the
colorimetric assay.
2?
K?, PO34 and Zn solubilization and siderophore
production Assays

The ability of the strains to solubilize potassium was

detected in a plate assay using mica (aluminum
potassium silicate, KAlSi3) as an insoluble potassium
salt at 0.2 % following as described previously (Hu et al.
2006). The phosphate and zinc solubilization abilities of
the isolated strains were tested on Pikovskayas agar
media (Pikovskaya 1948) supplemented with 0.15 %
Ca3(PO4)2 and 0.15 % ZnO, respectively as insoluble
nutrient forms. Strains able to dissolve these salts show a
transparent halo around colonies on the agar plate. A
qualitative siderophore production assay was performed
for each strain on plates supplemented with chrome
azurol S (CAS) as previously described (Schwyn and
Neilands 1987). The siderophore producer strains show
a color change in the CAS reagent from blue to orange.
Salt tolerance
To test the salt tolerance capacity of the newly isolated
strains, a 50 ll aliquot of a pre-inoculum TSB culture

123

(O.D600 = 0.8) was used to inoculate 50 ml 1 %

proteose peptone medium containing 0, 50, 100 or 200
NaCl. After 48 h, the growth was evaluated according
to the absorbance of the culture at 600 nm. Strains
were tested for their salt tolerance based on the relative
changes in their growth following the previously
published protocol (Siddikee et al. 2010). The highest
O.D. value obtained at different NaCl concentrations
was considered as a maximum salinity tolerance level
for each strain.
Gnotobiotic root elongation assay under normal
and saline conditions
ACC deaminase and IAA producer strains were tested
for gnotobiotic root elongation as described previously
(Penrose and Glick 2003). This assay is widely
accepted as a fast method to evaluate the effect of
bacterial strains on seedling root elongation. After
growing the strains in MM medium supplemented
with ACC for 72 h, canola (Brassica campestris)
seeds were coated with the selected strains, MgSO4
solution as negative or with Pseudomonas putida
UW4 (Duan et al. 2013) strain as a positive control.
The seeds were grown in growth pouches for 7 days at
28/25 C, 12/12 h day/night, weak light cycle before
the root measurements were taken. The ability of
selected strains to enhance root elongation under
saline conditions was tested by including a 100 mM
NaCl solution instead of the distilled water in the
growth pouches. Strains used in this assay were first
induced for ACC deaminase activity and salinity
tolerance by incubation at 32 C with shaking for 72 h
in MM medium containing ACC as the sole source of
nitrogen plus 50 mM NaCl before being used to coat
canola seeds.
Antibiotic resistance
Identified strains were tested for their ability to grow in
the presence of six different antibiotics on solid media
using the standard antibiotic working concentrations.
TSA plates separately supplemented with ampicillin
(50 lg ml-1), erythromycin (50 lg ml-1), kanamycin (50 lg ml-1), rifamycine (15 lg ml-1), streptomycin (50 lg ml-1), and tetracycline (15 lg ml-1)
were used in the assay. A fresh colony of each strain
growing on TSA medium was streaked on the TSA
supplemented with different antibiotics and incubated

Antonie van Leeuwenhoek

overnight at 32 C. The antibiotic concentrations were

selected based on the previously published protocol
(Rashid et al. 2012).
The GenBank/EMBL/DDBJ accession numbers of
the sequences reported in this paper are (KP259622
KP259707).

Results
Isolation of endophytic strains from plant roots
The microbial community structure and diversity was
not the focus of this study. Rather, individual endophytic strains were isolated and characterized. In this
project, two different strategies were used to isolate
endophytic bacteria from date palm roots. First, these
bacteria strains were extracted from the root tissues
using Ringers solution and then directly streaked on
rich agar media of different components regardless of
their ability to produce ACC deaminase. Second,
aliquots of the extracted Ringers solution were used as
inocula in selective media in sequential steps to isolate
ACC deaminase-producing bacterial strains. A total of

Table 1 Representative strains isolated from date palm roots.

Blast search results of the 16S ribosomal DNA sequences of
the isolated strains using the NCBI 16S rRNA gene collection
Strain

34 (group 1) and 51 strains (group 2) were isolated

using the first and the second strategies, respectively. A
sequence analysis of the 16S rDNA gene of group 1
showed that the first strategy led to the isolation of
bacteria strains related to the genera Bacillus, Chryseobacterium, Paenibacillus, Rhodococcus and Staphylococcus where Bacillus was dominant (Table 1,
Supplementary Table S1); however, the second
strategy led to the isolation of endophytes related to
the genera Achromobacter, Acinetobacter, Escherichia, Enterobacter and Klebsiella where Enterobacter
was dominant (Table 1, Supplementary Table S1).
Bacterial strains identified in this study were tested for
their ability to grow within plant tissues by inoculating
canola seeds with an individual bacterial isolate. The
results revealed that these strains have the ability to
grow internally in plant tissues and therefore they can
be considered as true endophytes.
Effect of salt concentration on ACC deaminase
activity
In this study, members of the newly isolated endophytes from the two isolated groups were able to

library. Gram stain result is demonstrated for each strain. Gram

staining procedure was repeated at least twice

Most similar strain

Gene Bank number of

the highest match

Identity (%)

Gram Stain

PD-R1

Rhodococcus equi strain DSM

NR_041910.1

PD-R3

Bacillus megaterium QM B1551 strain QM B1551

NR_074290.1

99.50

Positive

PD-R6

Paenibacillus xylanexedens strain B22a

NR_044524.1

97.76

Negative

PD-R10

Bacillus endophyticus strain 2DT

NR_025122.1

98.99

Positive

PD-R12

Bacillus oleronius strain ATCC

NR_043325.1

98.99

Negative

PD-R13
PD-R34

Paenibacillus glucanolyticus strain DSM 5162

Bacillus thuringiensis Bt407

NR_040883.1
NR_102506.1

98.49
98.99

Negative
Negative

PD-L4

Staphylococcus pasteuri strain ATCC51129

NR_028980.1

94.92

Positive

PD-L6

Bacillus anthracis strain Ames

NR_102499.1

99.25

Positive

Negative

Group 1
98.98

Positive

Group 1
PD-P8

Enterobacter cloacae subsp cloacae

NR_102794.1

98.98

PD-P11

Acinetobacter pittii

NR_117621.1

100.00

Negative

PD-P12

Achromobacter sp.

NR_025685.1

97.46

Negative

PD-P14

Escherichia sp.

NR_102804.1

96.51

Negative

PD-P33

Klebsiella oxytoca KCTC 1686 strain

NR_102982.1

99.24

Negative

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Antonie van Leeuwenhoek

Table 2 ACC deaminase activity (lmol mg-1 h-1), and IAA
and similar compounds (lg ml-1) produced by newly isolated
strains. Activity or product not detected in the assays is denoted
by N.D.
Strain

ACC-Deaminase

IAA and similar

compounds

Group 1
PD-R1
PD-R3

3.9
5.1

30.2
N.D.

PD-R6

12.5

25.9

PD-R10

13.5

N.D.

PD-R12

13.1

21.2

PD-R13

3.4

70.8

PD-R34

N.D.

78.1

PD-L4

N.D.

45.1

PD-L5

N.D.

101.5

PD-L6

3.3

N.D.

PD-P1
PD-P7

4.2
4.5

110.2
N.D.

PD-P8

11.2

N.D.

PD-P11

N.D.

N.D.

PD-P12

22.5

206.4

PD-P14

8.1

178.2

PD-P26

15.1

N.D.

PD-P33

26.6

N.D.

PD-P40

7.4

N.D.

PD-P42

10.5

N.D.

Group 2

produce ACC deaminase in different quantities. While

the majority (82 %) of group 2 strains were able to
produce ACC deaminase, fewer members (35 %) of
the group 1 strains had this capacity (Table 2 and
Supplementary Table S2). This efficiency difference
in the isolation of the ACC deaminase-producing
strains between groups 1 and 2 arose because the
group 2 strains were initially screened based on their
ability to utilize ACC as a sole source of nitrogen and
therefore had ACC deaminase activity.
The strains isolated in this study were tested for
their ability to grow under different salinity conditions
(0, 50, 100 and 200 mM NaCl). The results showed
that the majority of the strains could sustain the
addition of 100 mM NaCl to the TSB medium. At a
higher NaCl concentration, their growth rate started to
decline (Table 3, Supplementary Table S3). Strains
with consistent levels of ACC deaminase activity were

123

further tested for their ability to produce this enzyme

when the medium was supplemented with different
NaCl concentrations. The production of ACC deaminase was differentially affected by changes in NaCl
concentrations in the growth media. While salt showed
a negative effect on ACC activity in most tested
bacteria strains, 50 mM slightly induced the activity of
ACC deaminase in PD-P1, PD-P6 and PD-P10. Oneway analysis of variance (ANOVA) test showed that
this increase was only significant (p B 0.05) in strains
PD-P1 and PD-P6 (Fig. 1a). The O.D600 for the culture
was measured prior to the ACC deaminase activity
assays. The results did not show any consistent direct
relationship between the ACC deaminase activity and
the growth rate of the different strains (Fig. 1b). These
results may suggest the need for an elevated level of
NaCl in the microenvironment of these strains for
them to reach the optimum conditions for ACC
production, a situation that these strains may experience when date palms grow in saline conditions. In
comparison with Pseudomonas putida UW4, a rhizospheric, non-endophytic plant growth-promoting
strain which was used as a positive control, the strains
isolated in this study exhibited a somewhat lower level
of ACC activity; however, strain UW4 showed a
similar ACC production pattern to the majority of the
tested strains in response to increased NaCl levels in
the MM.
IAA-producing endophytes in the date palm
Several of the isolates analyzed in this study showed
the ability to produce different amounts of IAA or
similar compounds (Table 2 and Supplementary Table
S2) when grown in MM supplemented with tryptophan. The IAA contents were measured during the
exponential growth stage and before bacterial strains
entered into the stationary growth phase because it was
noticed that during stationary stage the free IAA
content dropped dramatically in the medium supplemented with tryptophan. Salinity alters growth rates
for the isolates and this may have a significant impact
on the measured IAA content. Therefore to avoid the
growth stage variation factor due to the treatments, the
IAA and similar molecules content were measured
when cells reached a specific density in the culture
(O.D.600 = 1).
To investigate the reason behind the IAA reduction
and to test whether these bacteria strains are

Antonie van Leeuwenhoek

Table 3 Maximum salt concentration tolerated by each strains
and their ability to produce ammonia, Fe3?-siderophores, and
to solubilize minerals. Strains with undetectable products or
Strain

Maximum salinity
(mM)

capacity to solubilize minerals are denoted by N.D., while

those with different levels of products or capacity to solubilize
minerals are denoted by a corresponding number of ? signs

Ammonia
production

Fe3?-siderophores
production

K? solubility

PO34 solubility

Zn2? solubility

N.D.

N.D.

N.D.

N.D.
N.D.

Group 1
PD-R1

50

PD-R3

N.D.

N.D.

N.D.

N.D.

PD-R6

150

N.D.

N.D.

N.D.

N.D.

N.D.

PD-R10

100

??

N.D.

N.D.

N.D.

PD-R12

100

N.D.

N.D.

N.D.

N.D.

PD-R13
PD-R34

100
100

N.D.
????

N.D.
N.D.

N.D.
N.D.

N.D.
N.D.

N.D.
N.D.

PD-L4

150

??

N.D.

N.D.

N.D.

N.D.

PD-L5

50

??

N.D.

N.D.

N.D.

N.D.

PD-L6

150

??

N.D.

N.D.

N.D.

N.D.

Group 2
PD-P1

100

N.D.

PD-P7

100

N.D.

N.D.

N.D.

N.D.

PD-P8

100

N.D.

N.D.

N.D.

N.D.

PD-P11

100

???

PD-P12

100

N.D.

N.D.

N.D.

PD-P14
PD-P26

100
200

?
?

N.D.
N.D.

N.D.
N.D.

N.D.
N.D.

N.D.
N.D.

PD-P33

100

N.D.

N.D.

PD-P40

100

??

N.D.

N.D.

N.D.

PD-P42

N.D.

N.D.

N.D.

N.D.

consuming IAA as a source of carbon and/or nitrogen,

MM lacking any source of nitrogen and/or carbon but
supplemented with 3 mM IAA were separately
inoculated with strains PD-P1, PD-P5, PD-P12, PDP15, PD-P18, PD-P19, PD-P37 and E. coli DH10B as
a negative control strain. After incubation at 32 C for
72 h, bacterial stains did not show any growth in these
media indicating the inability of these strains to use
IAA as a source of carbon or nitrogen during their
growth.
When these bacteria grew in MM in the absence of
tryptophan, the colorimetric assay did not show a
change in colour. In comparison with strain Pseudomonas putida UW4, the strains isolated in this study
had a greater capacity to produce IAA compounds.
The ability of these strains to produce IAA under
salinity stress was also tested for a selected group of
strains during the exponential growth stage (OD600 = 1)

since overgrowth may significantly reduce the detectable free IAA and similar compounds in the
supernatant. While salinity stress had a negative effect
on the majority of the tested strains in terms of IAA
production, some strains, such as PD-P5, PD-P15, PDP18 and PD-P37, were able to produce an additional
amount of IAA when grown in MM medium supplemented with tryptophan and 50 mM NaCl. ANOVA test
showed that this increase in IAA production was only
significant (p B 0.05) in strain PD-P15 and PD-P37
(Fig. 2).
Production of ammonia and siderophore
2?
and solubilization of K?, PO34 and Zn
All of the strains isolated in this study were tested for
their ability to produce ammonia in liquid proteose
peptone medium. A colorimetric assay showed that

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Antonie van Leeuwenhoek

ACC deaminase activity (mol mg1 h1)

Fig. 1 Endophytic
bacterial strains isolated
from date palm tree grown
under saline conditions and
the salt effect on the ACC
deaminase activity.
a Enzymatic assay showing
different levels of ACC
deaminase produced by
selected endophytes. Bars
represent mean SE
(n = 3). Significant
difference between the
control and the NaCl
treatment (p B 0.05) are
labeled by asterisk. b OD600
was measured for the
cultures prior to the ACC
deaminase activity assay.
Bacteria strains were
cultured in MM
supplemented with ACC as
a sole source of nitrogen

P 0.05
P 0.05

Optical Density (OD)

most of the bacterial strains had the ability to produce

different quantities of ammonia (Table 3, Supplementary Table S3). Since endophytes can provide plants
with other essential macro- and micronutrients, all of
the strains isolated in this study were tested for their
ability to chelate Fe3?, and to solubilize K?, PO34 and
Zn2?. The ability to dissolve these minerals was

123

determined by the production of a halo zone around

the colony growing in suitable solid medium in a Petri
dish. A few members of the group 1 strains showed the
ability to dissolve these minerals; however, a greater
number of group 2 strains demonstrated this ability. Of
these, one and 15 strains related to groups 1 and 2,
respectively, showed the ability to produce high-

Antonie van Leeuwenhoek

Fig. 2 Bacterial strains
isolated from date palm
response to saline conditions
and produce IAA at different
levels. Colorimetric assay of
IAA production by the
selected strains cultured in
MM media supplemented
with L-tryptophan.
Supernatant samples were
assayed during the
exponential growth phase
(OD600 = 1) of the culture.
Bars represent mean SE
(n = 3). Significant
difference between the
control and the NaCl
treatment (p B 0.05) are
labeled by asterisk

affinity iron-chelating siderophores (Table 3, Supplementary Table S3). In addition, four strains (PD-P2,
PD-P3, PD-P11 and PD-P12) were able to solubilize
K? from mica, 34 strains solubilized PO34 from the
insoluble Ca3(PO4)2 and 19 strains solubilized Zn2?
from the insoluble form of zinc oxide (ZnO) after
5 days of incubation at 32 C (Table 3, Supplementary Table S3).

Antibiotic resistance
The production of antibiotics is an important feature of
microbially-associated plant growth promotion since
antibiotic-resistant strains may have the ability to
outcompete other strains in the rhizosphere. All of the
bacterial strains in this study were tested for their
ability to resist growth inhibition by the antibiotics
ampicillin, erythromycin, kanamycin, rifamycine,
streptomycin and tetracycline using the regular antibiotic working concentrations. The results showed
that some strains from group 1 had the ability to only
resist ampicillin; however, all of the strains from
group 2 had the ability to resist different antibiotics
with the exception of rifamycine (Supplementary
Table S4). Therefore, the members of group 2 showed
a potentially broader range of competition against
other microbes living in the rhizosphere.

Canola root elongation enhancement

The gnotobiotic test was used to assess the effect of
some isolated bacterial strains on root elongation.
Strains with an ability to produce ACC deaminase and
IAA were selected and used to coat canola seeds. The
results showed that, in aggregate, these strains had the
ability to enhance canola root elongation by an
average of *30 % compared with the negative
control. An ANOVA test showed that this increase
in canola root length is significant (p B 0.05) in most
of the tested strains when compared with uncoated
seeds growing under the same conditions (Fig. 3). In
order to prove that these endophytes have the ability to
help plants growing under saline conditions and to
reduce the deleterious effect of salinity on growth, a
gnotobiotic root elongation assay was also carried out
using canola seeds growing under saline conditions
(100 mM NaCl). The saline conditions had a clear
negative effect on the canola seedlings as root
elongation growth was reduced in the control experiment by an average of 27 % (Fig. 4). Compared to
the seedlings growing in the control (distilled water)
experiment, some strains recovered their normal
growth rate and were also able to increase the canola
root length. There was no significant difference
(p B 0.05) in root length between uncoated seeds
growing under normal conditions and coated seeds

123

Antonie van Leeuwenhoek

Root Length (cm)

Fig. 4 Canola seeds treated with selected strains and tested for
their ability to enhance root elongation in the Gnotobiotic root
elongation assay. Seeds treated with 0.3 mM MgSO4 are used as
a negative control (negative) or coated with Pseudomonas
putida UW4 and used as a positive control (positive). Bars
represent mean SE (n = 24). Significant difference between
the bacteria treated seeds and the negative seeds growing under
100 mM NaCl conditions (p B 0.05) are labelled by asterisk.

123

* *
*

* * *

Root Length (cm)

Fig. 3 Endophytes
enhanced root elongation of
canola. Canola seeds treated
with selected strains and
tested for their ability to
enhance root elongation in
the Gnotobiotic root
elongation assay. Seeds
treated with 0.3 mM MgSO4
are used as a negative
control (negative). Bars
represent mean SE
(n = 24). Significant
difference between the
treated seeds and the
negative (p B 0.05) are
labeled by asterisk

Treatment with endophytic bacteria recovers the normal

phenotype when seeds grow under saline conditions. There is
no significant difference (p B 0.05) between negative treatment
growing under control condition and the bacteria-treated seeds
growing under 100 mM NaCl conditions. The horizontal dotted
line illustrates the differences in root elongation between the
salt-treated seedlings of the negative control and the salt and the
bacteria treated seedlings

Antonie van Leeuwenhoek

growing under saline conditions (Fig. 4) supporting

the hypothesis that these endophytes help plants grow
under saline conditions.

Discussion
Recently, the date palm tree has suffered from an
excessive accumulation of salt in soil due to anthropogenic activities. In this study, we isolated and
characterized members of the endophytic microbial
community in date palm seedling roots and studied
their effect on early plant growth under normal and
saline conditions.
In the date palm, the salinity tolerance mechanism is unknown, as is the role of endophytic
bacteria. Indeed, there is currently no information
available regarding the salt concentrations in the
apoplastic fluid in date palm roots although it can be
assumed that these concentrations are dependent on
the ability of the date palm roots to exclude or
absorb salt from the soil. Accordingly, this microenvironment determines the subsequent interaction of
endophytic strains and their effects on date palms
growing under saline conditions. However, the
bacterial strains isolated from date palm roots are
not expected to be halophilic because they are not
taken from salty soil. Therefore, their ability to
tolerate saline conditions is lower than that of other
identified halophilic strains (Siddikee et al. 2010).
In this paper, we investigated the role of a portion of
the endophytic bacterial community in date palm in
salinity tolerance. Based on the results obtained in this
study, there is some evidence consistent with the
possibility that these bacteria help date palms to grow
under saline conditions. First, some of the strains
isolated in this study were able to produce ACC
deaminase, which can cleave a portion of the ACC
produced as a consequence of salinity stress and
therefore reduce the amount of the stress hormone
ethylene that is produced (Table 2, Supplementary
Table S2, Fig. 1a). Ethylene is often overproduced in
plants as a result of a wide range of abiotic stresses,
including high salinity. By lowering ethylene levels,
the presence of ACC deaminase can reduce the
negative consequences of ethylene on plant growth
and development and increases plant tolerance to
salinity (Gamalero et al. 2009).

The present study also showed that the production

of ACC deaminase in a few strains is induced by
salinity (Fig. 1a). The positive role of growth-promoting ACC deaminase-producing bacteria in salinity
tolerance has been well established in several reports
(Ali et al. 2014; Cheng et al. 2007; Mayak et al. 2004a;
Siddikee et al. 2010, 2011); therefore, our results are
consistent with previously published works.
Second, some of the strains identified in this study
were able to produce the growth-promoting hormone
IAA (Table 2, Supplementary Table S2, Fig. 2). This
hormone has a profound effect on plant growth and
development, including the promotion of root formation. Additional amounts of NaCl in the growth
medium induced the production of IAA by some of
the strains tested in this study. This also may help
plants growing under saline conditions.
The IAA and similar compounds contents in the
growth media of some isolated strains in this study
were reduced when strains about to reach the stationary growth phase. According to our results, the reason
was not due to the consumption of IAA and similar
compounds by these strains as carbon or nitrogen
sources but presumably could be due to degradation,
mineralization (Faure et al. 2009) or conversion of free
IAA into IAA-lysine conjugate. In fact the amount of
the available IAA in bacteria depends on the production and the conversion rates of IAA (Duca et al. 2014)
and this is a strategy used by some bacteria strains to
adjust the amount of free IAA depending on bacterial
pathogenicity (Gomez-Manzo et al. 2010) and the host
demand for a healthy level of IAA (Sitbon et al. 1992).
This phenomenon is well known in other bacteria
species such as Pseudomonas savastanoi (Yamada
et al. 1985).
Some studies have shown that halophytes including strains of Bacillus subtilis, Brevibacterium halotolerans, Achromobacter xylosoxidans, Brachybacterium saurashtrense sp., and Pseudomonas sp.
that are also growth-promoting bacteria produce
IAA (Jha et al. 2012; Piccoli et al. 2011; Sgroy et al.
2009). In plants, auxin signalling and polar movement play an important role in root reorganization
and adaptation mechanisms in response to salinity
(Iglesias et al. 2010, 2011; Wang et al. 2009).
Depending on the plant and the conditions, extra
production of IAA has the potential to promote the
growth of the host plants.

123

Antonie van Leeuwenhoek

Salinity tolerance of the isolated strains was mainly

affected by the basic composition of the culture media
used. For example, some strains that showed tolerance
to a certain salt concentration in TSB media may not
show the same phenotype when grew in MM media
supplemented with ammonium acetate or ACC as sole
sources of nitrogen. In addition, the optimum salt
concentration for the growth of a particular strain may
not the optimum for IAA production and ACC
deaminase activity.
Third, some of these isolated bacterial strains were
able to produce ammonia and solubilize K?, Fe3?,
2?
PO3(Table 3, Supplementary Table S3).
4 and Zn
Plants consume macro- and micronutrients during
their routine growth and developmental processes;
however, under stress conditions the need for nutrients
is greater because plants are required to invest more in
order to meet the additional energy demands placed by
the physiological processes induced by salinity. Given
that the endophytes of date palms are able to produce
2?
ammonia and solubilize K?, Fe3?, PO34 and Zn ,
these endophytes may reduce the negative effects of
salinity and help plants to withstand stress conditions
by supplying the roots with an extra dose of nutrients.
Finally, some strains, regardless whether they
produced ACC deaminase or IAA, showed a positive
effect on root elongation when canola seeds were
grown under both normal and saline conditions
(Fig. 4). In fact, there is some evidence that both
ACC deaminase and IAA synthesis genes are under
the control of the stationary phase sigma factor so that
(up to a point) stress conditions may actually lead to an
increase in their synthesis (Patten and Glick 2002;
Saleh and Glick 2001).
Bacterial strains only producing IAA are able to
enhance root growth. For example, Pseudomonas
putida UW4 (AcdS-), although lacking the ACC
deaminase gene, is able to enhance root elongation
because of its ability to produce IAA (Patten and Glick
2002). In the present study, the addition of NaCl in the
gnotobiotic root elongation assay decreased root
elongation (Fig. 4); however, this decrease was
minimized in the presence of some of the newly
isolated strains. Given that some of these strains are
able to produce extra amounts of ACC deaminase and
IAA under higher salinity conditions, the enhancement of root elongation was not unexpected. In fact,
the positive effect of these strains on root elongation
under saline conditions suggests an explanation

123

regarding the involvement of these endophytic bacteria in the salt adaptation mechanisms of the date palm
tree. In this regard, assessment of the prevalence of
ACC deaminase among bacterial strains isolated from
the rhizosphere of wild barley growing in two adjacent
but very different micro-environments (Timmusk
et al. 2011) reported that under the more stressful
conditions, approximately 50 % of the isolated bacteria contained ACC deaminase while only approximately 4 % of the bacteria from the nonstressed environment contained this enzyme. The
stressed environment was sparsely vegetated as a
result of excessive sunlight and frequent drought while
the non-stressed environment had much more luxuriant plant growth and an absence of drought. These
results were interpreted as indicating that the stressful
environment selected for the presence of bacterial
ACC deaminase, protecting plants and facilitating
their survival. It should be noted that three of the
strains (PD-P6, PD-P7 and PD-P10) tested here using
the gnotobiotic assay (Fig. 4) didnt contribute significantly to canola root elongation under salinity
stress, where all of them presented ACC deaminase
activity and only one produced IAA (PD-10). It could
be speculated that more evident stress alleviation
comes from strains possessing several different plant
growth promoting abilities, in this case ACC deaminase and IAA production (Glick 2014). Interestedly,
only one of the strains (PD-P1) with increased ACC
deaminase under salinity conditions showed a significant canola root elongation under stress condition
(Fig. 1a).
In the present study we do not claim the identification of every endophytic strain in date palms,
however, it is our conclusion that some of the bacterial
strains isolated from date palm roots likely play a role
in salinity tolerance since they have the ability to
produce the growth regulator IAA and reduce ethylene
production through the production of ACC deaminase.
These strains are further able to provide the plant with
essential nutrients such as ammonia, K?, Fe3?, PO34
and Zn2?. However, the presence of other unidentified
mechanisms used by these bacteria to help plant
growth and development under salinity stress should
not be ignored.
Acknowledgments This work was supported by a generous
grant from the College of Science, Sultan Qaboos University IG/
Sci/Biol/13/01 to MWY. The authors would like to thank Prof.

Antonie van Leeuwenhoek

Jose A. Gil, University of Leon for his helpful comments on the
manuscript.
Conflict of interest
interest.

Authors do not claim any conflict of

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