Isolation and characterization of endophytic plant growth

ORIGINAL PAPER

Isolation and characterization of endophytic plant growth-promoting bacteria from date palm tree (Phoenix dactyliferaL.) 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 characteriza-

tion 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) dea-

minase and the plant growth regulatory hormone

indole-3-acetic acid (IAA). Some strains were also

able to chelate ferric iron (Fe3?) and solubilize

potassium (K?), phosphorus (PO43-) and zinc (Zn2?),

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

(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 Plant–microbe interactions �Endophytes � Date Palm � 1-aminocyclopropane-1-

carboxylic 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

Electronic supplementary material The online version ofthis article (doi:10.1007/s10482-015-0445-z) contains supple-mentary 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: [email protected]

B. R. Glick

Department of Biology, University of Waterloo,

Waterloo, ON N2L 3G1, Canada

123

Antonie van Leeuwenhoek

DOI 10.1007/s10482-015-0445-z

http://dx.doi.org/10.1007/s10482-015-0445-z

phosphate (PO43-) (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. Endo-

phytes 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 pro-

grammed 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-ketobu-

tyrate. 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 bacte-

ria’s importance is greater when plants are exposed to

environmental stress conditions. Under these condi-

tions, 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

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 Ringer’s solution as described (Rashid

et al. 2012). The eluted Ringer’s solutions were diluted

and 10 ll aliquots were spread onto King’s 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


123

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, 2–4 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 Ringer’s

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 hy-

drolysate, 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. Subse-

quently 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 endo-

phytes, 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-endo-

phytic 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 Labora-

tories) 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. Subse-

quently, 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 cen-

trifuged again at 5000 rpm for 30 min. The super-

natants 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-1L-tryptophan in MM to

produce IAA and similar molecules. To test the effect


123

of salinity on IAA production, MM was also supple-

mented 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 mea-

sured for all isolated strains as previously described

(Glickmann and Dessaux 1995) and using Salkowski’s

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 de-

scribed (Marques et al. 2010) by growing the bacteria

in 1 % proteose peptone at 32 �C for 24 h followed by

staining the culture with Nessler’s reagent in the

colorimetric assay.

K?, PO43- and Zn2?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 Pikovskaya’s 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

(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), kanamy-

cin (50 lg ml-1), rifamycine (15 lg ml-1), strepto-

mycin (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


123

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 endo-

phytic 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 Ringer’s 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 Ringer’s solution were used as

inocula in selective media in sequential steps to isolate

ACC deaminase-producing bacterial strains. A total of

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, Chry-

seobacterium, Paenibacillus, Rhodococcus and Sta-

phylococcus 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, Escheri-

chia, 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 endo-

phytes from the two isolated groups were able to

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

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

staining procedure was repeated at least twice

Strain Most similar strain Gene Bank number of

the highest match

Identity (%) Gram Stain

Group 1

PD-R1 Rhodococcus equi strain DSM NR_041910.1 98.98 Positive

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 Paenibacillus glucanolyticus strain DSM 5162 NR_040883.1 98.49 Negative

PD-R34 Bacillus thuringiensis Bt407 NR_102506.1 98.99 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

Group 1

PD-P8 Enterobacter cloacae subsp cloacae NR_102794.1 98.98 Negative

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


123

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

further tested for their ability to produce this enzyme

when the medium was supplemented with different

NaCl concentrations. The production of ACC deami-

nase 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. One-

way 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 experi-

ence when date palms grow in saline conditions. In

comparison with Pseudomonas putida UW4, a rhizo-

spheric, 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 trypto-

phan. 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 supple-

mented 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

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 3.9 30.2

PD-R3 5.1 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.

Group 2

PD-P1 4.2 110.2

PD-P7 4.5 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.


123

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, PD-

P15, 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 Pseu-

domonas 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 de-

tectable 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, PD-

P18 and PD-P37, were able to produce an additional

amount of IAA when grown in MM medium supple-

mented 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

and solubilization of K?, PO43- and Zn2?

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

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

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

Strain Maximum salinity

(mM)

Ammonia

production

Fe3?-siderophores

production

K? solubility PO43- solubility Zn2? solubility

Group 1

PD-R1 50 ? N.D. N.D. N.D. N.D.

PD-R3 0 N.D. 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 100 N.D. N.D. N.D. N.D. N.D.

PD-R34 100 ???? 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 100 ? N.D. N.D. N.D. N.D.

PD-P26 200 ? 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 0 N.D. ? N.D. N.D. N.D.


123

most of the bacterial strains had the ability to produce

different quantities of ammonia (Table 3, Supplemen-

tary 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?, PO43- and

Zn2?. The ability to dissolve these minerals was

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-

ACC

dea

min

ase

activ

ity (μ

mol

mg−

1h−

1 )

**

Opt

ical

Den

sity

(OD

)

A

B

P ≤ 0.05P ≤ 0.05

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


123

affinity iron-chelating siderophores (Table 3, Supple-

mentary 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 PO43- 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, Supplemen-

tary 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 an-

tibiotic 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 ex-

periment 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

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



control and the NaCl

treatment (p B 0.05) are

labeled by asterisk


123

Roo

t Len

gth

(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



treated seeds and the

negative (p B 0.05) are

labeled by asterisk

Roo

t Len

gth

(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.

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


123

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 anthro-

pogenic 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 mechan-

ism 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 microen-

vironment 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-pro-

moting 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 forma-

tion. 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 station-

ary 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 produc-

tion 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 includ-

ing strains of Bacillus subtilis, Brevibacterium halo-

tolerans, Achromobacter xylosoxidans, Brachy-

bacterium 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 move-

ment 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

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?,

PO43- and Zn2? (Table 3, Supplementary Table S3).

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

ammonia and solubilize K?, Fe3?, PO43- and Zn2?,

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 enhance-

ment of root elongation was not unexpected. In fact,

the positive effect of these strains on root elongation

under saline conditions suggests an explanation

regarding the involvement of these endophytic bacte-

ria 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 bac-

teria contained ACC deaminase while only ap-

proximately 4 % of the bacteria from the non-

stressed 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 luxuri-

ant 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) didn’t contribute sig-

nificantly 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 deami-

nase and IAA production (Glick 2014). Interestedly,

only one of the strains (PD-P1) with increased ACC

deaminase under salinity conditions showed a sig-

nificant canola root elongation under stress condition

(Fig. 1a).

In the present study we do not claim the identifi-

cation 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?, PO43-

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.


123

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

manuscript.

Conflict of interest Authors do not claim any conflict of

interest.

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