Halophytes-associated endophytic and 
rhizospheric bacteria: diversity, antagonism 
and metabolite production
Authors: Fehmida Bibi, Gary A. Strobel, Muhammad 
Yasir, Ahmed Abdulah Khalaf, and Esam Ibrahim 
Azhar
This is an Accepted Manuscript of an article published in Biocontrol Science and Technology on 
February 15, 2018, available online: 
htp:/www.tandfonline.com/10.1080/09583157.2018.1434868.
Bibi, Fehmida, Gary Alan Strobel, Muhammad Imran Naseer, Muhammad Yasir, Ahmed Abdulah 
Khalaf Al-Ghamdi, and Esam Ibrahim Azhar. "Halophytes-associated endophytic and rhizospheric 
bacteria: diversity, antagonism and metabolite production." Biocontrol Science and Technology 
28, no. 2 (February 2018): 192-213. DOI: 10.1080/09583157.2018.1434868.
Made available through Montana State University’s ScholarWorks 
scholarworks.montana.edu 
Halophytes-associated endophytic and rhizospheric bacteria:
diversity, antagonism and metabolite production
Fehmida Bibi a, Gary Alan Strobelb, Muhammad Imran Naseerc, Muhammad Yasira,
Ahmed Abdulah Khalaf Al-Ghamdidand Esam Ibrahim Azhara,d
aSpecial Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi
Arabia;bDepartment of Plant Sciences, Montana State University, Bozeman, MT, USA;cCenter of Excelence in
Genomic Medicine Research (CEGMR), King Abdulaziz University, Jeddah, Saudi Arabia;dDepartment of
Medical Laboratory Technology, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah,
Saudi Arabia
ABSTRACT
In Saudi Arabia, halophytes occupy tidal and intertidal forest
ecosystems. They and their associated microflora have immense
potential to yield novel and important useful natural products.
Three halophytes (Avicennia marina,Halocnemum strobilaceum,
Zygophylum qatarense) were targeted for the isolation and
identification of populations of endophytic and rhizospheric
bacteria having antimicrobial potential. A total 554 bacterial
isolates were initialy screened against oomycetes fungal
pathogens,Phytophthora capsiciandPythium ultimum. Of these,
only 57 rhizospheric and endophytic bacteria exhibited inhibition
against the targeted bioassay oomycetes.Tentative identification
of the bacteria was on the basis of 16S rRNA gene sequences
which revealed 92–100% sequence identity to type strains of
related species and placed these organisms in six major classes:
Actinobacteria, γ-Proteobacteria, Firmicutes, α-Proteobacteria,
Flavobacteria and β-Proteobacteria. When checked for lytic
enzyme production, mostly the isolates ofActinobacteriaand
Firmicutes were potential enzyme producers. Detection of
secondary metabolite biosynthetic genes–type I polyketide
synthases, type I polyketide synthases and nonribosomal peptide
synthetases–confirmed that 21 (35.5%) isolates were positive for
at least one type of the biosynthetic gene. In order to identify
metabolites, three isolates, Alteromonas australica (EA73),
Aidingimonas halophila (EA105) andHalomonas zincidurans
(EA127), were selected and subjected to chemical analyses using
liquid chromatography–mass spectrometry and gas
chromatography–mass spectrometry. Both analyses showed the
presence of diferent bioactive compounds in the culture extracts
of isolates some of which are already reported for their diverse
biological activities such as 2, 4-Diacetylphloroglucinol. Our results
demonstrated that halophytes represent an important source of
potentialy active bacteria producing antifungal metabolites of
medical significance.
Introduction
Halophytes inhabit the intertidal areas of Saudi Arabia and they consist of populations of
grasses, epiphytes, shrubs and trees. Among halophytes, mangroves exist in tropical cli-
mates requiring warm conditions and can tolerate salinity, anaerobic conditions, tides,
winds and high temperatures. Their habitat enables them to grow with rich biodiversity.
Mangroves under these stressful conditions are able to produce diferent kinds of active
metabolites with diverse biological functions. Until now, more than 200 active metabolites
have been isolated from mangroves (Bandaranayake,2002).
Mangroves are a good source of steroids, saponins, flavonoids, triterpenes, alkaloids,
tannins and have been used in diferent traditional medicines due to the antimicrobial
activities of their extracts (Eldeen & Efendy,2013). Previous studies reported a wide
range of biological activities such as antiviral, antibacterial and antifungal properties of
plant extracts (Chandrasekaran, Kannathasan, Venkatesalu, & Prabhakar,2009; Prema-
nathan et al.,2009). Due to their biological activities, they are an ideal material for inves-
tigation of associated microorganisms and their bioactive compounds. Both rhizospheric
and endophytic bacteria seem to play important roles for the survival and growth of host
plants. They provide several beneficial efects including plant growth promotion and
resistance against pathogens by producing diferent substances such as volatile and anti-
fungal compounds (Chung et al.,2008; Ryu et al.,2003). Halophytes also harbour diferent
groups of bacteria, and these bacteria perform diferent functions such as growth pro-
motion and disease resistance against diferent pathogens (Thatoi, Behera, Mishra, &
Duta,2013). Bacterial communities associated with halophytes are beneficial for the
host whether as rhizospheric bacteria (Roy, Hens, Biswas, Biswas, & Kumar,2002)or
in endophytic form (Garcias-Bonet, Arrieta, de Santana, Duarte, & Marbà,2012;Hu,
Li, & He,2010). These bacteria are also an excelent source of diferent useful enzymes
and antibiotics (Thatoi et al.,2013).
Many halophytes have associated endophytic bacteria that possess a broad spectrum of
antifungal activities against diferent pathogenic bacteria (Hu et al.,2010; Jose & Christy,
2013). Recently, new marine compounds such as isocoumarin, furanocoumarin and kan-
denol have been reported from halophyte-associated bacteria (Xu,2015); however, there
are limited studies on isolation of bacteria from halophytes in coastal areas of the Red
Sea and their associated identification and biological activities. In a recent study from
Saudi Arabia, microbial communities from a mangrove (Avicennia marina) were ident-
ified using a metagenomic approach but no functional aspects of the microbes involved
have been studied (Alzubaidy et al.,2016). Therefore, we designed a study to isolate
both rhizospheric and endophytic bacteria from diferent parts of halophytes,A.
marina, Halocnemum strobilaceumandZygophylum qatarense, and to perform a tentative
identification of the strains using 16S rDNA molecular techniques and screened them
against diferent phytopathogens. We also report enzyme production, secondary metab-
olite biosynthetic genes and the identification of metabolites from selected bacterial iso-
lates. Three potential bacteria were selected for identification of metabolites and
antimicrobial compounds such as 2,4-Diacetylphloroglucinol (DAPG), prednicarbate,
benzydamine, methyl jasmonate, cephaloridine and triamcinolone acetonide were
discovered.
Materials and methods
Sample colection and isolation of rhizospheric bacteria from halophytes
Three diferent halophytes specimens [A. marina(mangrove),H.strobilaceumandZ.
qatarense)] were colected from the coastal area of Thuwal, Jeddah, Saudi Arabia (22°
15′54′North, 39° 6′44′East). Al of the plant specimens were placed in a sterile bag
after colection and transferred to the laboratory for bacterial isolation. Soil, roots,
leaves and pneumatophores of halophytes were used for the isolation of rhizospheric
and endophytic bacteria. To isolate bacteria from soil adhering to roots of the
plants,theyweredippedinsteriledistiledwaterandserialdilutionswere made
(10−3,10−4and 10−5)infilteredautoclavedseawater(FAS)andspreadintriplicates
on four diferent media used for culturing of bacteria. Half strength R2A (½ R2A)
[0.25 g yeast extract; 0.25 g Proteose Peptone No. 3 (Difco Laboratories, Detroit,
MI); 0.25 g casamino acid; 0.25 g dextrose;0.25 g soluble starch; 0.15 g sodium pyru-
vate; 0.15 g K2HPO4;0.03gMgSO4], half strength Tryptic soy agar (½ TSA) [pancrea-
tic digest of casein 7.5 g; papain digestof soybean 2.5 g; NaCl 2.5 g; agar 15.0 g],
marine agar (MA) [peptone 5.0 g; yeast extract 1.0 g; Ferric Citrate 0.1 g; NaCl
19.45 g; MgCl 8.8 g; Na2SO43.24 g; CaCl 1.8 g; KCl 0.55 g; NaHCO30.16 g; KBr;
0.08 g; SrCl234.0 mg; H3BO322.0 mg; Na2SiO34.0 mg; NaF 2.4 mg; NH4NO3
1.6 mg; Na2HPO48.0 mg; agar15.0 g] and half nutrient agar (½ NA) [beef extract
1.5 g; peptone 2.5 g; agar 15.0 g] (Difco Laboratories) for bacterial culturing in
1000 mL filtered seawater for bacterial culturing. Roots and leaves tissues were also
used for isolation of bacteria.
Isolation of endophytic bacteria from halophytes
For isolation of endophytic bacteria from plant roots, leaves and pneumatophores surface
sterilisation was performed. Roots, leaves and pneumatophores segments were washed 5–6
times with tap water and then further sterilised by washing with disinfectants as described
previously (Bibi, Yasir, Song, Lee, & Chung,2012). After sterilisation of roots, leaves and
pneumatophores segments, smal pieces of sterilised roots, leaves and pneumatophores
segments were ground in FAS using sterile mortar and pestle. Aliquots were further seri-
aly diluted (10−3,10−4and 10−5) and plated in triplicate on the four diferent media men-
tioned above. To suppress fungal growth, 50μg/mL cycloheximide was mixed to the
medium before pouring. Plates were incubated at 25°C for 2 weeks for bacterial growth.
Individual colonies were further streaked to check the purity of the strains, and al
these bacterial strains were further subcultured and stored in 15% (v/v) glycerol stock
of strains at−70°C in King Fahd Medical Research Centre and were given lab numbers
from EA73-EA131, respectively.
Screening of bacteria for antifungal activity
Rhizospheric and endophytic bacteria isolated from halophytes were further screened for
their antifungal potential. Five diferent fungal pathogens:Phytophthora capsici
(P. capsici),Pythium ultimum (Py. ultimum), Magnaporthe grisea(obtained in this labora-
tory),Altenaria mali(KCTC 6972) andFusarium moniliforme(KCTC 6149), were
obtained from the Korean type culture colection centre (KCTC).Antagonistic activity
against fungal pathogens was determined by using the cross streak method (Bibi et al.,
2012). Al isolates were streaked on PDA media supplemented with ½ R2A in seawater.
Each 6-mm mycelial disc of test fungal pathogens was placed in the centre of the plate per-
pendicular to the streak of isolates at 4 cm from the edges of the plate and was incubated
for 4–6 days at 28°C. Al strains were checked twice for antagonistic activity. The antag-
onistic activity was then evaluated by measuring the inhibition zone of fungal mycelia
around the bacterial colony.
Extraction of bacterial DNA and 16S rRNA gene sequencing
Genomic DNA was extracted from antagonistic bacteria using a DNA extraction kit
(Thermo Scientific, Waltham, MA, USA). To identify antagonistic bacteria, 16S rRNA
gene sequencing was performed. Using bacterial universal primers 27F (5′-AGAGTTT-
GATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT -3′), the 16S
rRNA gene fragment was amplified. Amplifications were performed under folowing con-
ditions: one cycle of 95°C for 5 min folowed by 28 cycles of 95°C for 1 min, an annealing
of 58°C for 50 s and extension at 72°C for 1 min, with a final extension step at 72°C for
10 min. Using PCR purification kit (Thermo Scientific), the PCR products were purified
and sequenced commercialy by Macrogen (Seoul, Korea). Sequences obtained after 16S
rRNA gene similarity were identified using the EzTaxon server (htp:/eztaxon-e.
ezbiocloud.net/) (Kim et al.,2012) to identify the bacteria. To determine the phylogenetic
position of the antagonistic bacteria and related type strains, the 16S rRNA gene sequences
of type strains were obtained from the National Center for Biotechnology Information.
For the phylogenetic analysis, CLUSTALX (Thompson, Gibson, Plewniak, Jeanmougin,
& Higgins,1997) multiple alignments of the bacterial sequences were performed and
BioEdit software (Hal,1999) was used to edit the gaps. The neighbour-joining method
in the MEGA6 Programme with bootstrap values based on 1000 replications was used
for constructing the phylogenetic tree (Tamura, Stecher, Peterson, Filipski, & Kumar,
2013).
Evaluation of hydrolytic enzymatic activity
Protease activity was checked using skim milk ½ R2A agar plates. Bacteria producing
protease made clear zone on skim milk agar plates. Amylase production was checked
on starch media. Amylase producing bacteria showed starch hydrolysis as a clear
zone on starch ½ R2A agar plates (Kumar, Karan, Kapoor, Singh, & Khare,2012).
For lipase activity, tributyrin ½ R2A agar media was used. After 48 h of incubation
at 28°C, a clear zone was detected around bacteria that were hydrolysing tributyrin.
To check celulase activity, CMC agar (carboxymethylcelulose agar) media was used.
Bacteria were streaked on plates of the respective enzyme media and incubated at
28°C for 2 days. After these plates were flooded with a solution of 0.1% Congo red
and put on an orbital shaker for 15 min and washed with 1M NaCl (Hendricks,
Doyle, & Hugley,1995). Positive activity was seen as a halo zone around bacterial colo-
nies on CMC agar.
Detection of secondary metabolite biosynthetic genes
The detection of a biosynthetic gene, polyketide synthetase I (PKS-I) and nonribosomal
peptide synthetase (NRPS) genes were checked using primer pairs K1F/M6R (5′-
TSAAGTCSAACATCGGBCA-3′;5′-CGCAGGTTSCSGTACCAGTA-3′) and A3F/A7R
(5′GCSTACSYSATSTACACSTCSGG-3′;5′-SASGTCVCCSGTSCGGTAS-3′), respect-
ively (Ayuso-Sacido & Geniloud,2005). The polyketide synthetase I (PKS-I) gene was
amplified using primer pair KSα/Kβ (5′-TSGCSTGCTTGGAYGCSATC-3′; 5′-
TGGAANCCGCCGAABCCTCT-3′) (Metsä-Ketelä et al.,1999). PCR amplification pro-
ducts were then checked by using 0.8% agarose gel electrophoresis, and bands of 1.2–
1.4 kb, 600 bp and 700–800 bp were detected as products of PKS-I, PKS-I and NRPS
genes, respectively.
Optimisation of bacterial culture condition for production of antifungal activity
To optimise culture conditions of the selected bacterial strains for the production of anti-
fungal activities, an appropriate medium for culturing was selected. Four diferent media,
i.e. ½ R2A broth, ½ TSA, ½ NA in seawater and Marine broth in distiled, were used for
culturing. After every 24 h, optical density (OD) was checked and antifungal activity was
assessed againstP. capsiciandPy. ultimumusing disc difusion method. The efect of
temperature was checked at diferent ranges of temperatures (20–40°C) in ½ R2A
broth. For pH optimisation, diferent pH value ranges (5–12) were used for the growth
and antifungal compound production in ½ R2A broth.
Gas chromatography–mass spectrometry analysis for identification of
metabolites
Al chemicals and solvents were of MS grade.N,O-bis(trimethylsily) trifluoractamide + tri-
methylchlorosilane (TMCS) (99:1), Methoxyamine hydrochloride and Pyridine (R.G.,
Reag. ACS, Reag. Ph Eur) were purchased from Sigma-Aldrich, and methanol was pur-
chased from Thermo Fisher. Bacterial cultures (1 mL) were lysed and centrifuged at
10,000gfor 10 min. The supernatant (200 µL) was transferred to a 1.5-mL Eppendorf
tube and 600 µL acetonitrile was added and vortex for 30 s. Samples were further centri-
fuge at 13,000gfor 10 min and 200 µL supernatant was transferred to glass vials. The
solvent was removed by a nitrogen dry machine. Further 80 µL methoxy amine pyridine
hydrochloride (15 mg/mL) was added to the glass-derived botle, vortexed for 2 min and
incubated at 80°C for 30 min;N,O-bis(trimethylsily) trifluoractamide (BSTFA) 80μL con-
taining 1% (v/v) TMCS was added to each vial and the mixture was vortexed for 2 min and
then derivatised at 80°C for 30 min. Mixtures were centrifuged for 10 min (13,000g), then
100μL supernatant was used for gas chromatography–mass spectrometry (GC-MS) meta-
bolomics analysis. The derivatised samples were analysed on Shimadzu GCMS-QP2010
Ultra, including a capilary column (30 m × 0.25 mm × 0.25μm) used to separate the
derivatives. Helium (>99.999%) was used as the carrier gas at a constant flow rate of
1 mL/min through the column. The injector temperature was maintained at 260°C and
injection volume was 1μL in spoutless mode. The initial oven temperature was 80°C
and was held for 2 min, ramped to 300°C at a rate of 10°C/min, and finaly held at
300°C for 5 min. The temperature of ion source (electron impact) was set to 200°C. The
colision energy was 70eV. Mass data were acquired in a ful-scan mode (m/z45–800),
with an acquisition rate of 100 spectrum/s.
Liquid chromatography–mass spectrometry analysis of bacterial culture
Five mililitres of bacterial culture was placed on−80°C for 5 min and then transferred to a
37°C water bath for 5 min, and this procedure was repeated for five times. Centrifugation
was done at 15,000gfor 10 min, 3 mL supernatant was transferred to a tube and 12 mL
acetonitrile was added and vortexed for 30 s. Again, centrifugation was done at 15000g
for 10 min and 300 µL supernatant was taken for liquid chromatography–mass spec-
trometry (LC-MS) metabolomics analysis. Injection volume was 3 µL and the samples
are analysed on Agilent 6540B TOF/Q-TOF Mass Spectrometer coupled with Agilent
1290 UPLC and Dual AJS ESI ion source. An ACQUITY UPLC HSS T3 (100 × 2.1 mm,
1.8μm) column and pre-column (Phenomenex Security Guard™) was used to separate
sample. The column temperature was set to 45°C and the flow rate was 0.5 mL/min.
Acquisition range was from 50 to 1500m/z, and scan rate was 1.00 spectrum/s. MS par-
ameters were set as folows: capilary voltage 3500 V, nebulizer pressure 35 psi, drying gas
10 L/min, gas temperature 325°C, vaporizer 200 V, voltage charge 1000 V; negative-ion
mode capilary voltage 3500 V, corona negative 15.0 V, fragmentor 175 V, skimmer1
65.0 V, octopole RF Peak 750 V; positive ion mode capilary voltage 3500 V, corona posi-
tive 4.0 V, fragmentor 175 V, skimmer1 65.0 V and octopole RF Peak 750 V. Raw data
were imported to Agilent MassHunter Qualitative Analysis B.06.00 software. Metabolites
were identified by the in-house database.
Nucleotide sequence numbers
Al of the nucleotide sequences of the bacterial isolates from this study have been deposited
in the GenBank database under accession numbers KY436446–KY436504.
Statistical analysis
Metabolites were identified by the NIST database. Briefly, after alignment with Statistic
Compare component, the CSV file was obtained with three-dimensional data-sets includ-
ing sample information, peak name, retention time,m/zand peak intensity. Internal stan-
dard peak and peaks caused by noise, column bleed and BSTFA derivatisation procedure,
were removed from the data-set, and the peaks from the same metabolites were combined.
Results
Isolation of rhizospheric and endophytic bacteria from halophytes
In this study, three halophytes were colected from the coastal area of Thuwal, Jeddah (Fig.
S1) and were identified asA. marina(mangrove), H. strobilaceumandZ. qatarense(Fig.
S2(a–d)). The diversity of rhizospheric and endophytic was evaluated from soil, roots,
leaves and pneumatophores of halophytes. A total of 554 colony-forming rhizospheric
and endophytic bacteria were isolated from these three halophytes (Table 1). We have
used five diferent culturing media for isolation of rhizopheric and endophytic bacteria
were used. These diferent media that are in high and low nutrient contents favour difer-
ent groups of bacteria to grow. Some of the bacterial colonies were so smal and required
2–3 weeks to grow.
Screening of bacteria against pathogenic fungi
Rhizospheric and endophytic bacteria were tested for their antagonism against pathogenic
oomycetes,Py. ultimumandP. capsici. Of 554 bacteria, 57 (10.2%) displayed inhibition to
both oomycete plant pathogens. The proportion of antagonistic bacteria varied, being
highest inA. marina(n= 28; 12.3%) andZ. qatarense(n= 16; 11.8%), folowed byH. stro-
bilaceum(n= 13; 6.7%) (Table 1). These antagonistic bacteria were then tested for other
three fungal pathogens. Diferent groups of antagonistic rhizospheric and endophytic bac-
teria were identified from halophytes, whereγ-Proteobacteriawas the dominant phylum
(Table 1). Antagonistic activity of al bacterial isolates is summarised inTable 2. Some
strains showed strong inhibition with 7–9 mm against oomycetes pathogens. Among 57
antagonistic bacteria, only 4 (6.7%),Alteromonassp. (EA73)Streptomycessp. (EA91),
Bacilussp. (EA115) andHalomonassp. (EA127), exhibited inhibitory activity against
al pathogenic fungi tested, whereasBacilussp. (EA78),Bacilussp. (EA81),Mycobacter-
iumsp. (EA84),Bacilussp. (EA98) andHalomonassp. (EA127) had strong inhibition
only against oomycetes (Table 2). Some antagonistic bacteria were active against only
oomycetes and were inactive for other pathogenic fungi used for screening in this study
(Table 2).Halomonaswas the dominant genus in this study among 30 diferent genera
of antagonistic bacteria isolated from halophytes.
Phylogenetic analysis of antagonistic bacteria based on 16S rRNA gene sequence
Antagonistic bacteria (n= 57) were identified by using 16S rRNA gene sequence analysis.
Twenty diferent genera of bacteria were identified and belonged to five major classes:
Actinobacteria(n= 12; 21%),γ-Proteobacteria(n= 24; 42%),Firmicutes(n= 10; 18%),
α-Proteobacteria(n= 8; 14%), andFlavobacteria(n=3; 5%) (Figure 1).The proportion
of diferent generaineach part of plant sample and soil is shown inFigure 1(b–e).
Sequence identity of antagonistic bacteria was from 92.8% to 100% (Table 2). The phylo-
genetic tree inferred using 16S rRNA gene data showed that branching paterns remained
constant. High bootstrap values were recorded in the phylogenetic tree using 16S rRNA
Table 1.Distribution of rhizospheric and endophytic antagonistic bacteria isolated from halophytes.
Plant speciesa Isolatesb Antagonistsс Antagonistsd(%) Dominant phylume
Avicennia marina 226 28 12.3 Actinobacteria
Halocnemum strobilaceum 193 13 6.7 γ-Proteobacteria
Zygophylum qatarense 135 16 11.8 γ-Proteobacteria
554 57 10.3 γ-Proteobacteria
aScientific names of halophytes colected from coastal area for isolation of bacteria.
bTotal number of bacteria isolated from halophytes.
сTotal number of antagonistic bacteria againstP. capsiciandPy. ultimumby confrontation bioassay.
dPercentage of antagonistic bacteria from total bacteria.
eDominant phylum in al antagonistic bacteria to oomycetes.
Table 2.Taxonomic identification, antifungal activity and enzymes production of rhizospheric and endophytic bacteria from halophytes.
Antifungal activity againsta Enzymatic activitiesb Detection ofc
Lab
no Closely related type straind
Accession
number
%
identitye
Py.
ultimum P. capsici M. grisea A. mali F. moniformeProtease Amylase Lipase Celulase NRPS
PKS-
I
PKS-
I
Avicennia marina
Rhizopheric bacteria (Soil)
EA73 Alteromonas australicaH17T KY436446 98.6 +++ ++ + +++ − − − ++++
+
− − − −
EA74 Alteromonas macleodiATCC 27126T KY436447 98.7 + + − −  −  −  − ++++
+
− − − −
EA75 Alteromonas australicaH17T KY436448 97.3 ++ + − −  −  −  − ++++
+
− − − −
EA76 Pseudoalteromonas flavipulchra
NCIMB 2033T
KY436449 97.7 ++ + − −  −  −  − − − − − −
EA77 Halomonas smyrnensisAAD6T KY436450 99.7 + + − −  −  −  − − − − − −
EA78 Bacilus licheniformisATCC 14580T KY436451 99 +++ +++ ++ − − +++++ +++++ − − − − −
EA79 Halomonas denitrificansM29T KY436452 99.4 + +++ − −  −  −  − − − − − −
EA80 Alteromonas gracilis9a2T KY436453 92.8 ++ + − −  −  −  − − − − − −
EA81 Bacilus persicusB48T KY436454 97.2 +++ +++ − −  −  −  − − − − − −
Endophytic bacteria (Pneumatophores)
EA82 Devosia subaequorisHST3-14T KY436455 98.3 ++ W − −  −  −  − ++++ − − − −
EA83 Nocardioides aromaticivoransH-1T KY436456 99.4 + + − −  − ++++ − ++++ − + − −
EA84 Mycobacterium wolinskyiATCC
700010T
KY436457 99.9 +++ +++ − −  −  −  − ++ ++++ + − −
EA85 Streptomyces spectabilisNBRC 13424T KY436458 95.9 + ++ − −  −  −  − − − + + +
EA86 Erythrobacter citreusRE35F/1T KY436459 98.2 W + − −  −  −  − − − − − −
EA87 Nocardioides albusKCTC 9186T KY436460 99.1 ++ + − −  − +++++
+
− ++++ − +
EA88 Halomonas denitrificansM29T KY436461 99.2 + ++ + − −  − − − − − − −
EA89 Nocardioides luteusKCTC 9575T KY436462 100 + + − −  − +++++
+
− +++ − − − −
EA90 Marinobacter mobilisCN46T KY436463 98.3 + ++ + − −  − − − − − − −
EA91 Streptomyces enissocaesilisNBRC
100763T
KY436464 100 ++ ++ ++ +++ ++ +++ − + − + + +
Endophytic bacteria (Roots)
EA94 Nocardioides luteusKCTC 9575T KY436467 100 ++ W − −  −  −  − − − − − −
EA95 Nocardioides albusKCTC 9186T KY436468 96.8 W + + − −  − − − − + − −
EA96 Nitratireductor shengliensis110399T KY436469 97.4 ++ + + ++ − − − − − − − −
EA97 Inquilinus limosusDSM 16000T KY436470 96.4 +++ + + − −  − − − − − − −
EA98 Bacilus beringensisBR035T KY436471 97.5 +++ +++ − −  −  −  − − − − − −
EA99 Amycolatopsis thermalbaSF45T KY436472 98.9 + + ++ − −  − − − − + − +
Endophytic bacteria (Leaf)
EA100 Micrococcus flavusLW4T KY436473 99.8 + + − −  −  −  − − − − − −
EA101 Brevibacterium caseiNCDO 204T KY436474 99.3 W + − −  −  −  − − − + − +
EA102 Staphylococcus epidermidisATCC
14990T
KY436475 99.5 ++ + − −  −  −  − − − − − −
Halocnemum strobilaceum
Rhizopheric bacteria (Soil)
EA103 Oceanicola litoreusM-M22T KY436476 96.3 + + − −  −  −  − +++ − − + −
EA104 Roseisalinus antarcticusEL-88T KY436477 94.1 + + − −  −  −  − − − − − −
EA105 Aidingimonas halophilaYIM 90637T KY436478 97.8 +++ + ++ ++ − − − +++ − − − −
EA106 Halomonas anticariensisFP35T KY436479 97.7 ++ ++ ++ − −  − − − − + + +
EA107 Halomonas luteaDSM 23508T KY436480 99 ++ +++ + − −  − − +++ − − − −
EA108 Halomonas smyrnensisAAD6T KY436481 99.8 + ++ − −  −  −  − − − − − −
Endophytic bacteria (Roots)
EA109 Marinobacter lacisalsiFP2.5T KY436482 98.2 ++ + − −  −  −  − − − + − −
EA110 Bacilus timonensisMM10403188T KY436483 96.9 + ++ + + − − − − − + − −
EA111 Halomonas luteaDSM 23508T KY436484 96.9 + ++ W − −  − − − − − − −
EA112 Salipiger mucosusDSM 16094T KY436485 99.6 ++ +++ + + − − − − − − − −
EA113 Rubrimonas cliftonensisOCh317T KY436486 92.9 + + + − −  − − − − + − −
Leaf
EA114 Staphylococcus hominis subsp.
novobiosepticusGTC 1228T
KY436487 99.7 ++ ++ − −  −  −  − − − − − −
EA115 Bacilus subtilis subsp. inaquosorum
KCTC 13429T
KY436488 99.9 +++ ++ + + + − − − − − − +
Zygophylum qatarense
Rhizopheric bacteria (Soil)
EA116 Muricauda lutimarisSMK-108T KY436489 97.6 + ++ + − −  − ++ ++++ − − − −
EA117 Tamlana crocinaHST1-43T KY436490 99.4 + + − −  − ++ − − − − − −
EA118 Marinimicrobium haloxylanilyticum
SX15T
KY436491 96.9 + ++ W − −  − − − − − − −
EA119 Marinobacter salsuginisSD-14BT KY436492 97.3 ++ +++ + − − ++ − − − + + −
EA120 Halobacilus trueperiDSM 10404T KY436493 99.4 +++ +++ − −  −  −  − − − − − −
EA121 Zunongwangia mangroviP2E16T KY436494 98.9 + + − −  −  −  − − − − − +
Endophytic bacteria (Roots)
EA122 Marinobacter mobilisCN46T KY436495 97.9 + ++ ++ − −  − ++ ++++ − + − +
EA123 Halomonas smyrnensisAAD6T KY436496 100 + + + − −  − − − − + − −
EA124 Marinobacter lacisalsiFP2.5T KY436497 96.5 + ++ − −  −  − + − + + −
(Continued)
Table 2.Continued.
Antifungal activity againsta Enzymatic activitiesb Detection ofc
Lab
no Closely related type straind
Accession
number
%
identitye
Py.
ultimum P. capsici M. grisea A. mali F. moniformeProtease Amylase Lipase Celulase NRPS
PKS-
I
PKS-
I
++++
+
EA125 Microbulbifer celerISL-39T KY436498 99.3 ++ W − −  −  −  − ++ − + − +
EA126 Bacilus licheniformisATCC 14580T KY436499 99.3 ++ +++ ++ + − +++++ + +++ − − − −
EA127 Halomonas zinciduransB6T KY436500 96.7 +++ +++ + + +++ − − − − − − −
Endophytic bacteria (Leaf)
EA128 Arthrobacter crystalopoietesDSM
20117T
KY436501 97.3 + + − −  −  −  − − − − − −
EA129 Halomonas smyrnensisAAD6T KY436502 99.7 + + + − −  − − − − + − −
EA130 Staphylococcus warneriATCC 27836T KY436503 99.8 ++ ++ − −  −  −  − +++ − − − −
EA131 Marinobacter algicolaDG893T KY436504 97.7 ++ ++ ++ − −  − − +++ − + − −
aAntagonistic activity of al bacteria isolated in this study. The activity was measured after 3–5 days incubation at 28°C by measuring the clear zone of mycelial growth inhibition:−, Negative; +,
3 mm; ++, between 4 and 6 mm; +++, between 7 and 9 mm
bProduction of protease, amylase, lipase and celulase was determined by plate assay. Enzymatic activity was estimated as zone of halo formed around bacterial colonies:−, Negative; +, 3 mm; ++,
between 4 and 6 mm; +++, between 7 and 9 mm; ++++, between 10 amd 12 +++++, between 13 and 15.
cDetection of NRPS and PKS-I and PKS-I genes in antagonistic bacteria.
dIdentification based on partial 16S rRNA gene sequence analyses of al antagonistic bacteria.
e% similarity with closely related type strain
gene sequences data (Figure 2). Antagonistic strains of classFirmicuteswere placed in a
separate cluster recovered with higher bootstrap values. Antagonistic bacteria inFirmi-
cutesmainly belonged to the generaBacilus,StaphylococcusandHalobacilus. Three
diferent clusters have been generated for isolates of classActinobacteria. Antagonistic
bacteria in these three clusters were identified with high bootstraps values (54–100%).
Representative isolates in this class belong to four diferent genera, i.e.Nocardioides,
Arthrobacter, StreptomycesandMycobacterium. Three strains ofFlavobacteriawere
identified with a separate cluster also showing high bootstrap values (64–100%). The
strains ofα-Proteobacteriacomprising eight diferent genera were placed in one cluster
in the phylogenetic tree with high bootstrap values. The representative strains ofγ-Proteo-
bacteriabelong to five diferent genera. Al strains of the genusHalomonasmake a distinct
cluster with the closely related type strainHalomonas denitrificansM29Twith a bootstrap
value of 99–100%. In this study, some novel and new antagonistic rhizospheric and endo-
phytic bacterial strains were also identified in addition to common and already known
bacteria (Table 2). Some antagonistic rhizospheric and endophytic bacteria were recovered
showing low 16S rRNA gene sequence similarity (<95%). Two isolates ofα-Proteobacteria,
i.e.Roseisalinussp. (EA104) andRubrimonassp. (EA113), and one isolate belonging toγ-
Proteobacteria, i.e.Alteromonassp. (EA80), were identified as novel species with simi-
larities of <95% to the closest type strains in GenBank (Table 2). In addition to these
novel isolates, many other novel antagonistic rhizospheric and endophytic bacteria
Figure 1.(a) Percentage composition of diferent phyla of antagonistic rhizospheric and endophytic
bacteria isolated from four halophytes on the basis of 16S rRNA gene sequence similarity. Percentage
composition of diferent phyla in (b) soil, (c) roots, (d) leaves and (e) pneumatophores.
belonging to diferent genera were identified in this study. More potential antagonistic
candidates have been identified from the classγ-Proteobacteria. Some strains ofγ-Proteo-
bacteriawith high antagonistic activity against pathogenic fungi were common and had a
sequence similarity of 100% with the relative type strain.Alteromonassp. (EA73),Aidin-
gimonassp. (EA105) andHalomonassp. (EA127) and had moderate to strong inhibitory
activity and were identified as novel strains with sequence similarity of 98.6%, 97.8% and
96.7%, respectively, with relative type strains. We have selected these three antagonistic
strains depending on their 16S rRNA sequence similarity and antagonistic activity for sec-
ondary metabolite identification.
Enzymatic activities of antagonistic bacteria
Antagonistic rhizospheric and endophytic bacteria were evaluated for their ability to
produce cel wal lytic enzymes. We have tested protease, amylase, lipase and celulase
activities (Table 2). The number of antagonistic rhizospheric and endophytic bacteria
exhibiting lipase activity (n= 19; 32%) was high as compared to other enzymatic activities.
Figure 2.Phylogenetic distribution of antagonistic bacteria isolated from halophytes on the basis of
16S rRNA gene sequences obtained from bacteria and closely related sequences of the type strains
of other species. The phylogenetic relationships were infered from the 16S rRNA gene by using the
neighbour-joining method from distances computed with the Jukes–Cantor algorithm. Bootstrap
values (1000 replicates) are shown next to the branches. GenBank accession numbers for each
sequence are shown in parentheses. Bar represents 0.01 accumulated changes per nucleotide. Isolates
selected for bioactive metabolites identification are highlighted.
Most of lipase-producing bacteria were endophytes (n= 17) and were isolated from leaves
and root tissues of halophytes. Three rhizospheric bacteria, strains EA73–EA75 belonging
toAlteromonas, displayed high lipase activity (Table 2). Protease production was observed
lower (n= 8; 13.5%) than lipase activity. Most of the endophytes (n= 5) were potential
protease enzyme producers. MostlyActinobacteriaandFirmicutesstrains were recorded
for high protease production. Strains ofBacilussp. (EA78),Nocardioidessp. (EA87),
Nocardioidessp. (EA89) andBacilussp. (EA126) had strong protease production.
Amylase activity was recorded for 6 (10%) antagonistic bacteria. Most of the bacterial
strains with strong amylase activity are species of the classFirmicutes. Endophytic
strain ofBacilussp. (EA78) exhibited high production of amylase. Celulase production
was observed in only 1 (1.6%) bacterial strains. A strain ofMycobacteriumsp. (EA84)
exhibited celulase production and it belongs toActinobacteria. Some antagonistic
strains were negative (n= 24; 40.6%) for production of al tested enzyme activities.
Detection of NRPS and PKS-I systems
Al antagonistic bacterial isolates were subjected to genetic screening for the presence of
NRPS and PKS genes. Of 57 isolates tested, PCR amplification revealed that 21 strains
(36.8%)werepositiveforatleastoneofthebiosyntheticgenetested(Table 2). And
for the NRPS gene, 18 (31%) strains were positive. Most of the strains were positive
for NRPS genes and belonged to the classActinobacteria.ForthegenePKS-I,only7
(12%) antagonistic bacterial strains were positive. Similarly, PKS-II gene cluster was
detected in only 9 (15.2%) strains. Both these genes PKS-I and PKS-II were mostly
detected in the strains ofActinobacteria. Three bacterial isolates,Erythrobactersp.
(EA85),Streptomycessp. (EA91) andHalomonassp. (EA106), in this study possess al
of the biosynthetic genes (NRPS, PKS-I and PKS-II). Two isolates belonging toγ-Proteo-
bacteria,Marinobactersp. (EA119) andMarinobactersp. (EA124), showed the presence
of both NRPS and PKS-I genes. Similarly, two isolates ofActinobacteria,Amycolatopsis
sp. (EA99) andBrevibacteriumsp. (EA101), were positive for the presence of both NRPS
and PKS-II system.
Culture condition optimisation and identification of metabolites by GC-MS and
LC-MS
In al tested media, ½ R2A broth was found to be the best culturing media. Selected
strains showed the best growth at pH 7.5 and 28°C in the shaking incubator
(140 rpm). After optimisation of conditions, selected bacterial strains were grown
in 5 mL ½ R2A broth in seawater for 36–48 h until OD600reached 0.9. From these
57 antagonistic bacteria, only 3 antagonistic isolates were selected belonging to the
classγ-Proteobacteria,i.e.Alteromonassp. (EA73),Aidingimonassp. (EA105) and
Halomonassp. (EA127) due to their low sequence similarity and high antifungal
activity. GC-MS and LC-MS analyses of culture of these three strains identified
diferent chemical constituents (Figures 3(a–c) and4(a–e)). For GC-MS, diferent
chemical compounds were identified by comparing their mass spectra with the
NIST library. IsolateAlteromonassp. (EA73) exhibited the presence of bioactive com-
pounds like aminoguanidine, styramate, azetidine and cyacetacide (Figure 3(a))
which have been already known for their use in the pharmaceutical industry (Har-
rington,2011). The culture extract of strainAidingimonassp. (EA105) showed the
presence of chemical compounds of biological significance such as benzopyrazine,
benzofurazan, isoborneol, betamethasone borneol and thalidomide valerate (Figure
3(b)). Chemical analyses ofHalomonassp. (EA127) resulted in the identification of
diferent active metabolites namely silanamine, cephaloridine and oxiraneundecanoic
(Figure 3(c)).
LC-MS analysis was performed to identify metabolites from culture extract. LC-MS
analysis ofAlteromonassp. (EA73) identified peaks for only nine secondary metabolites
in both the positive- and negative-ion mode (Figure 4(a,b)). These compounds include
DAPG, methyl jasmonate, prednicarbate, methoxamedrine, benzydamine, 5-formylsa-
licylic acid, geranylgeraniol, stearamide and erucamide. ForAidingimonassp. (EA105),
26 diferent peaks were identified where 4 active compounds including alopurinol, trigo-
neline, triamcinolone acetone and dodecanedioic acid were detected in the negative-ion
mode (Figure 4(c)).Halomonassp. (EA127) showed the presence of four bioactive com-
pounds in their culture extract, i.e. DAPG, benzydamine and dodecanedioic acid (Figure 4
(d,e)). In this study, the antibiotic DAPG is produced by two strainsAlteromonassp.
(EA73) andHalomonassp. (EA127) that is already known for its antiphytopathogenic
and biocontrol properties.
Figure 3.Bioactive compounds detected by GC-MS analysis. (a) GC-MS spectra ofAlteromonassp.
(EA73), (b)Aidingimonassp. (EA105) and (c)Halomonassp. (EA127).
Discussion
Halophytes exist under extreme environmental conditions which lead to the production of
certain metabolites enabling them to survive under these harsh conditions (De Carvalho &
Fernandes,2010). In this study, we have isolated 57 diferent antagonistic bacteria active
against diferent common fungal pathogens. For the isolation of bacteria, we used four
Figure 4.Spectra of LC-MS analysis showing detection of various bioactive metabolites.Alteromonas
sp. (EA73) (a) Positive mode LC-MS analysis and (b) negative mode LC-MS analysis; (c) Negative mode
Aidingimonassp. (EA105) LC-MS analysis; (d)Halomonassp. (EA127) Positive mode and (e) negative
mode LC-MS analysis.
diferent types of media in low nutrient concentration which results in the isolation of
diverse groups of antagonistic bacteria. We have also used this methodology in our pre-
vious studies and this has resulted in the isolation of many novel isolates with unique
bioactivities (Bibi et al.,2012,2017). To our knowledge, this is the first study on the iso-
lation, screening, characterisation and metabolite identification of rhizospheric and endo-
phytic bacteria from halophytes in Saudi Arabia.
In this study, we have used a culture-dependent approach to explore the diversity of
antagonistic rhizospheric and endophytic bacteria. Screening of 554 rhizospheric and endo-
phytic bacteria against oomycetes and identification on the basis of 16S rRNA gene sequence
resulted in the identification of 57 diferent bacteria comprising five major classes. Bioactive
compounds production by rhizospheric and endophytic bacteria is one of the techniques
used by bacteria to defend the host against diferent pathogens (Coombs, Michelsen, &
Franco,2004). Several previous studies have reported the production of antimicrobial com-
pounds by bacteria isolated from mangrove plants (Eldeen & Efendy,2013; Hu et al.,2010).
We have isolated a high number of species of antagonistic rhizospheric and endophytic bac-
teria from these plants. High percentage of antagonistic bacteria may be resulted as diferent
culturing media, isolation source (soil, roots and leaves) and only oomycetes pathogens as
target organisms were used for screening. Some previous studies reported the final
limited outcome of bacterial diversity when only soil was used as a microbial isolation
source (Mitra, Santra, & Mukherjee,2008;Rameshkumar&Nair,2009). Most of the antag-
onistic bacteria were recovered from the root (n= 27; 46%) tissue samples. Similarly, the
Figure 4.Continued
number of antagonistic bacteria was higher in soil (n= 23; 39%) as compared to leaf tissues
(n=9;15%)(Figure 1(b–d). The importance of endophytes from mangrove plants has been
previously reported (Eldeen & Efendy,2013; Menpara & Chanda,2013), and in our study,
endophytes exhibited strong antagonistic activities against diferent fungal pathogens. These
bacterial endophytes belong to genusBacilusand can be considered as potential candidates
for use as biocontrol agents.
Identification of these antagonistic bacteria based on the 16S rRNA gene resulted in five
major classes of bacteria. Phylogenetic analysis yielded a tree topology that grouped al antag-
onistic rhizospheric bacteria and endophytic into five diferent groups:Actinobacteria
(Nocardioides,Arthrobacter,Streptomyces,Mycobacterium),γ-Proteobacteria(Alteromonas,
Pseudoalteromonas,Halomonas,Marinobacter,Aidingimonas), Firmicutes(Bacilus,Staphy-
lococcus,Halobacilus),α-Proteobacteria(Devosia,Erythrobacter,Nitratireductor,Inquilinus,
Oceanicola,Aidingimonas,Salipiger,Rubrimonas), andFlavobacteria (Muricauda,
TamLana, Zunongwangia)(Figure 1and 2). Some genera were represented by several
species in these halophytes while some other genera were represented by only single
species. Our results demonstrated that classγ-Proteobacteriawas dominant (41%) among
al the antagonistic bacterial communities isolated from four diferent halophytes (Tables 1
and2). In this study, five diferent genera ofγ-Protobacteria(Pseudomonas,Alteromonas,
Pseudoalteromonas,Halomonas,Marinobacter,Aidingimonas) were isolated. Among
Gram-negative marine bacteria,γ-Proteobacteriaproduced the highest number of secondary
metabolites with functional diversity (Long & Azam,2001). In this study, we also choose iso-
latesAlteromonassp. (EA73),Aidingimonassp. (EA105) andHalomonassp. (EA127) belong
toγ-Proteobacteriafor secondary metabolite identifications.
The second dominant class of bacteria wasActinobacteria(20%) andFirmicutes(20%).
Today, the marine actinomycetes are considered as a major source for discovery of novel
natural products. MarineActinobacteriaare a source of novel antimicrobial compounds,
many of them such as salinosporamides are in clinical trials for use as anticancer agents
(Fenical & Jensen,2006). Previously, diferent strains ofActinobacteriaisolated from man-
grove sediments showed antimicrobial activity against diferent human pathogenic bac-
teria (Lee et al.,2014). In this study, most of the antagonistic rhizospheric and
endophytic bacteria belonging to the classActinobacteriain this study such asNocar-
dioides, Arthrobacter, Pseudonocardia, Streptomyces, AgromycesandMycobacteriumare
already known for their antagonism and production of antimicrobial metabolites
(Fenical & Jensen,2006). Bacteria belonging to classFirmicutesare commonly found
both as rhizospheric and endophytic bacteria as they are easily cultivated. In marine
environments, strains ofBacilusare the dominant producers of antibacterial, antifungal
antibiotics, enzymes and surfactants (Mondol, Shin, & Islam,2013). The representative
ofFirmicutesin our study belonged toBacilus, StaphylococcusandHalobacilusspecies
are already known to produce antifungal substances (Wagner-Döbler, Beil, Lang,
Meiners, & Laatsch,2002). Previously two endophytic bacterial strains ofBaciluswere
isolated from mangrove plants and showed inhibition against bacterial and fungal plant
pathogens (Menpara & Chanda,2013). In this study, 14% antagonistic rhizospheric and
endophytic bacteria were related to classα-Proteobacteriaincluding eight diferent
genera.Several strains ofα-Proteobacteriaisolated from marine sources live in symbiosis
and nitrogen fixers, and produce antibiotics (Castro et al.,2014; Wagner-Döbler et al.,
2002). In this study, three strains ofFlavobacteriawere isolated from soil samples
adhering roots of host halophyte,Z. qatarense. Two antagonistic strains ofFlavobacteria,
Muricaudasp. (EA116) andMarinimicrobiumsp. (EA118) seems novel based on their 16S
rRNA similarity to related type strains.
Marine bacteria are already known for the production of diferent hydrolytic enzymes
exhibiting diverse enzymatic activities and catalysing diferent biochemical processes
using these enzymes (Thatoi et al.,2013). Most of the bacteria in our study produced
lipase, protease and amylase enzymes. Only two strains were found positive for production
of celulase. MostlyActinobacteriaandFirmicutesstrains were able to produce diferent
enzymes.Bacilussp. (EA126) showed protease, amylase and lipase activity. This strain
is endophytic and isolated from roots of halophyteZ. qatarense. These hydrolytic
enzymes are necessary for antagonism as wel as for intracelular colonisation of bacteria
to host plant. Castro et al. (2014) have evaluated mangrove plants for isolation and screen-
ing of bacteria for enzymatic activities. They found diferent groups of endophytic enzy-
matic bacteria whereFirmicuteswas the dominant class consistent with our study.
Marine bacteria possess PKS and NRPS genes which are halmarks for the production
of secondary metabolites. Of 57 isolates tested for PKS-I, PKS-I and NRPS gene, only 12%
were positive for PKS-I, 15% for PKS-I and 31% for NRPS genes. Mostly strains ofActi-
nobacteriawere positive for the presence of PKS-I and PKS-I genes. Two endophytic iso-
lates,Streptomycessp. (EA85) andStreptomycessp. (EA91), and one rhizospheric bacteria,
Halomonassp. (EA106), possess al of the biosynthetic genes (NRPS, PKS-I and PKS-I)
tested. The PCR-based detection of PKS-I, PKS-I and NRPS genes is important in screen-
ing and identification of bacterial isolates capable of producing active secondary metab-
olites. In our study, some isolates including three strains chosen for chemical analysis
were negative for amplification of these genes and may be due to the use of degenerate
primers which were not suitable for these genes (Qin et al.,2009; Qin, Li, Dastager, &
Hozzein,2016; Salomon, Magarvey, & Sherman,2004). Detection of PKS-I and PKS-I
genes was high in strains ofActinobacteria, providing a confirmation for the high possi-
bility ofActinobacteriaproducing active secondary metabolites.
The antifungal potential of selected isolates was influenced by culturing in R2A as
culture media using optimum culture conditions. The maximum antifungal activity was
observed after 48 h of growth at 28°C with pH 7.5. Three selected Gram-negative bacteria
belong to classγ-Proteobacteriawere analysed using LC-MS and GC-MS for secondary
metabolites present in their culture extract. We used LC-MS in addition to GC-MS as
LC technique mainly focuses on polar metabolites (especialy phosphate-containing com-
pounds), most of which cannot be analysed using GC-MS. Both analyses confirm the pres-
ence of various bioactive compounds, although not novel but already known for their
bioactivity. The antibiotic DAPG was detected in two isolatesAlteromonassp. (EA73)
andHalomonassp. (EA127) culture extracts when analysed by LC-MS. DAPG is an anti-
biotic with broad-spectrum antibacterial and antifungal activities produced byPseudomo-
nas fluorescensstrain that has been used as a biocontrol agent against several plant
pathogens includingPy. ultimum(Delany et al.,2000) which is also a test pathogen in
our study. Both these strains,Alteromonassp. (EA73) andHalomonassp. (EA127),
showed strong inhibition againstPy. ultimumin anin vitroassay when we tested. Detec-
tion of DAPG in both these strains suggests its role in these bacteria as an antifungal agent
against tested pathogens. Members of genusAlteromonasusualy associated with marine
sponges produce compounds such as a macrolactam and amide ester with cytotoxic and
antimicrobial activity (Thomas, Kavlekar, & LokaBharathi,2010). Similarly, strains of
Halomonasfrom a marine source are able to produce siderophore, Loihichelins (Giddings
& Newman,2015). But no previous study has reported the production of DAPG by marine
strains of these two genera.Alteromonassp. (EA73) also showed the presence of some
anti-inflammatory compounds such as prednicarbate and benzydamine are used as
drugs where benzydamine has antibacterial activity (Fanaki & El-Nakeeb,1992). In
addition to these compounds, methyl jasmonate, volatile compounds produced by
plants as a defence mechanism (Gimenez-Ibanez, Chini, & Solano,2016), were also
detected inAlteromonassp. (EA73) culture extract. Aminoguanidine and styramate
have medical uses in diabetic and anticonvulsant drugs, respectively, were also detected.
No such compound has been reported previously in strains ofAlteromonas.
Cephaloridine is a semisynthetic cephalosporin antibiotic, and was detected inAidingi-
monassp. (EA105). This derivative of cephalosporin C is used in respiratory tract infections.
This is first report of cephaloridine detection in bacteria.Aidingimonassp. (EA105) was also
able to produce benzydamine in addition to triamcinolone acetonide and dodecanedioic
acid. Triamcinolone acetonide is synthetic corticosteroid and used in various skin infections
while dodecanedioic acid has various industrial uses. LikeAidingimonassp. (EA105), triam-
cinolone acetonide and dodecanedioic acid also were detected inHalomonassp. (EA127).
Furthermore,Halomonassp. (EA127) also exhibited production of alopurinol and thalido-
mide valerate which have their use in pharmaceutical industry. According to LC-MS and
GC-MS analyses, al compounds detected in these three selected strains have their medicinal
use and are reported as anti-inflammatory, antibacterial and antifungal compounds. These
compounds seem to be responsible for antifungal activities of these three strains.
Conclusions
In this study, 57 antagonistic rhizospheric and endophytic bacteria inhabiting halophytes
have been isolated and partialy characterised. Among al, 21 bacterial strains have at least
one type of biosynthetic gene cluster, indicating their importance and potential for discov-
ery of new bioactive compounds. Moreover, these antagonistic bacteria exhibited enzy-
matic activities indicating their industrial and biotechnological potential. Analyses of
selected potential strains exhibited the presence of various bioactive metabolites including
known antibiotics and bioactive compounds of synthetic nature not reported from bac-
teria before. These results suggest that halophytes are a potential source of unexplored
metabolites. Antagonistic bacteria from halophytes growing in the coastal area of the
Red Sea in Saudi Arabia have the potential to produce a diverse range of antimicrobial
compounds that can be used in medicine and as a biocontrol agent in agriculture.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This project was funded by the National Plan for Science, Technology and Innovation (MAARI-
FAH)–King Abdulaziz City for Science and Technology, the Kingdom of Saudi Arabia (award
number 12-BIO-2724-03). The authors also acknowledge with thanks the Science and Technology
Unit, King Abdulaziz University for technical support.
ORCID
Fehmida Bibi htp:/orcid.org/0000-0002-5178-1471
References
Alzubaidy, H., Essack, M., Malas, T. B., Bokhari, A., Motwali, O., Kamanu, F. K.,…Archer, J. A. C.
(2016). Rhizosphere microbiome metagenomics of gray mangroves (Avicennia marina) in the
Red Sea.Gene,576, 626–636.
Ayuso-Sacido, A., & Geniloud, O. (2005). New PCR primers for the screening of NRPS and PKS-I
systems in actinomycetes: Detection and distribution of these biosynthetic gene sequences in
major taxonomic groups.Microbial Ecology,49,10–24.
Bandaranayake, W. M. (2002). Bioactivities, bioactive compounds and chemical constituents of
mangrove plants.Wetlands Ecology and Management,10, 421–452.
Bibi, F., Ulah, I., Alvi, S. A., Bakhsh, S. A., Yasir, M., Al-Ghamdi, A. A. K., & Azhar, E. I. (2017). Isolation,
diversity, and biotechnological potential of rhizoand endophytic bacteria associated with mangrove
plants from Saudi Arabia.Genetics and Molecular Research,16(2).doi:10.4238/gmr16029657
Bibi, F., Yasir, M., Song, G. C., Lee, S. Y., & Chung, Y. R. (2012). Diversity and characterization of
endophytic bacteria associated with tidal flat plants and their antagonistic efects on oomycetous
plant pathogens.The Plant Pathology Journal,28,20–31.
Castro, R. A., Quecine, M. C., Lacava, P. T., Batista, B. D., Luvizoto, D. M., Marcon, J.…Azevedo,
J. L. (2014). Isolation and enzyme bioprospection of endophytic bacteria associated with plants of
Brazilian mangrove ecosystem.SpringerPlus,3, 382.
Chandrasekaran, M., Kannathasan, K., Venkatesalu, V., & Prabhakar, K. (2009). Antibacterial
activity of some salt marsh halophytes and mangrove plants against methicilin resistant
Staphylococcus aureus.World Journal of Microbiology and Biotechnology,25, 155–160.
Chung, B. S., Aslam, Z., Kim, S. W., Kim, G. G., Kang, H. S., Ahn, J. W., & Chung, Y. R. (2008). A
bacterial endophyte,Pseudomonas brasicacearumYC5480, isolated from the root of Artemisia
sp. producing antifungal and phytotoxic compounds.The Plant Pathology Journal,24, 461–468.
Coombs, J. T., Michelsen, P. P., & Franco, C. M. (2004). Evaluation of endophytic actinobacteria as
antagonists ofGaeumannomyces graminisvar.triticiin wheat.Biological Control,29,359–366.
De Carvalho, C.C., & Fernandes, P. (2010). Production of metabolites as bacterial responses to the
marine environment.Marine Drugs,8, 705–727.
Delany, I., Sheehan, M. M., Fenton, A., Bardin, S., Aarons, S., & O’Gara, F. (2000). Regulation of
production of the antifungal metabolite 2,4-diacetylphloroglucinol in pseudomonas fluorescens
F113: Genetic analysis of phlF as a transcriptional repressor.Microbiology,146, 537–546.
Eldeen, I. M., & Efendy, M. A. (2013). Antimicrobial agents from mangrove plants and their endo-
phytes. In A. Méndez-Vilas (Ed.),Microbial pathogens and strategies for combating them: Science,
technology and education(pp. 872–882). FORMATEX Microbiology Book Series. Badajoz:
Formatex Research Centre.
Fanaki, N. H., & El-Nakeeb, M. A. (1992). Antimicrobial activity of benzydamine, a non-steroid
anti-inflammatory agent.Journal of Chemotherapy,4, 347–352.
Fenical, W., & Jensen, P. R. (2006). Developing a new resource for drug discovery: Marine actino-
mycete bacteria.Nature Chemical Biology,2, 666–673.
Garcias-Bonet, N., Arrieta, J. M., de Santana, C. N., Duarte, C. M., & Marbà, N. (2012). Endophytic
bacterial community of a Mediterranean marine angiosperm (Posidonia oceanica).Frontiers in
Microbiology,3,1–16.
Giddings, L. A., & Newman, D. J. (2015). Bioactive compounds from marine extremophiles. In
Bioactive compounds from marine extremophiles (pp. 1–124). Heidelberg: Springer
International Publishing.
Gimenez-Ibanez, S., Chini, A., & Solano, R. (2016). How microbes twist jasmonate signaling around
their litle fingers.Plants,5,9.
Hal, T. (1999). Bioedit: A user-friendly biological sequence alignment editor and analysis program
for Windows 95/98/NT.Nucleic Acids Symposium Series,41,95–98.
Harrington, P. J. (2011).Pharmaceutical proces chemistry for synthesis: Rethinking the routes to
scale-up. Hoboken, NJ: John Wiley & Sons.
Hendricks, C. W., Doyle, J. D., & Hugley, B. (1995). A new solid medium for enumerating celulose-
utilizing bacteria in soil.Applied and Environmental Microbiology,61, 2016–2019.
Hu, H. Q., Li, X. S., & He, H. (2010). Characterization of an antimicrobial material from a newly
isolatedBacilus amyloliquefaciensfrom mangrove for biocontrol of capsicum bacterial wilt.
Biological Control,54, 359–365.
Jose, A. C., & Christy, P. H. (2013). Assessment of antimicrobial potential of endophytic bacteria
isolated fromRhizophora mucronata.International Journal of Current Microbiology and
Applied Science,2, 188–194.
Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H.,…Won, S. (2012). Introducing
EzTaxon-e: A prokaryotic 16S rRNA gene sequence database with phylotypes that represent
uncultured species.International Journal of Systematic and Evolutionary Microbiology,62,
716–721.
Kumar, S., Karan, R., Kapoor, S., Singh, S. P., & Khare, S. K. (2012). Screening and isolation of halo-
philic bacteria producing industrialy important enzymes.Brazilian Journal of Microbiology,43,
1595–1603.
Lee, L. H., Zainal, N., Azman, A. S., Eng, S. K., Goh, B. H., Yin, W. F., & Chan, K. G. (2014).
Diversity and antimicrobial activities of actinobacteria isolated from tropical mangrove sedi-
ments in Malaysia.Scientific World Journal,2014, 698178.
Long, R. A., & Azam, F. (2001). Antagonistic interactions among marine pelagic bacteria.Applied
and Environmental Microbiology,67, 4975–4983.
Menpara, D., & Chanda, S. (2013).Endophytic bacteria-unexploredreservoir of antimicrobials for
combating microbial pathogens. InFORMATEX microbiology series 4–Microbial pathogens and
strategies for combating them: Science, technology and education(pp. 1095–1003). Badajoz:
Formatex Research Center.
Metsä-Ketelä, M., Salo, V., Halo, L., Hautala, A., Hakala, J., Mäntsälä, P., & Ylihonko, K. (1999). An
eficient approach for screening minimal PKS genes fromStreptomyces.FEMS Microbiology
Leters,180,1–6.
Mitra, A., Santra, S. C., & Mukherjee, J. (2008). Distribution of actinomycetes, their antagonistic
behaviour and the physico-chemical characteristics of the world’s largest tidal mangrove
forest.Applied Microbiology and Biotechnology,80, 685–695.
Mondol, M. A., Shin, H. J., & Islam, M. T. (2013). Diversity of secondary metabolites from marine
Bacilus species: Chemistry and biological activity.Marine Drugs,11, 2846–2872.
Premanathan, M., Arakaki, R., Izumi, H., Kathiresan, K., Nakano, M., Yamamoto, N., &
Nakashima, H. (2009). Antiviral properties of a mangrove plant,Rhizophora apiculataBlume,
against human immunodeficiency virus.Antiviral Research,44, 113–122.
Qin, S., Li, J., Chen, H. H., Zhao, G. Z., Zhu, W. Y., Jiang, C. L.,…Li, W. J. (2009). Isolation, diver-
sity, and antimicrobial activity of rare actinobacteria from medicinal plants of tropical rain
forests in Xishuangbanna, China.Applied and Environmental Microbiology,75, 6176–6186.
Qin, S., Li, W. J., Dastager, S. G., & Hozzein, W. N. (2016). Actinobacteria in special and extreme
habitats: Diversity, function roles, and environmental adaptations.Frontiers in Microbiology,7,
1415.
Rameshkumar, N., & Nair, S. (2009). Isolation and molecular characterization of geneticaly diverse
antagonistic, diazotrophic red-pigmented vibrios from diferent mangrove rhizospheres.FEMS
Microbiology Ecology,67, 455–467.
Roy, S., Hens, D., Biswas, D., Biswas, D., & Kumar, R. (2002). Survey of petroleum-degrading bac-
teria in coastal waters of Sunderban Biosphere Reserve.World Journal of Microbiology and
Biotechnology,18, 575–581.
Ryu, C. M., Farag, M. A., Hu, C. H., Reddy, M. S., Wei, H. X., Paré, P. W., & Kloepper, J. W. (2003).
Bacterial volatiles promote growth inArabidopsis.Proceedings of the National Academy of
Sciences of the United States of America,100, 4927–4932.
Salomon, C. E., Magarvey, N. A., & Sherman, D. H. (2004). Merging the potential of microbial gen-
etics with biological and chemical diversity: An even brighter future for marine natural product
drug discovery.Natural Product Reports,21, 105–121.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: molecular evol-
utionary genetics analysis version 6.0.Molecular Biology and Evolution,30, 2725–2729.
Thatoi, H., Behera, B. C., Mishra, R. R., & Duta, S. K. (2013). Biodiversity and biotechnological
potential of microorganisms from mangrove ecosystems: A review.Annals of Microbiology,
63,1–19.
Thomas, T. R., Kavlekar, D. P., & LokaBharathi, P. A. (2010). Marine drugs from sponge-microbe
association—A review.Marine Drugs,8, 1417–1468.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., & Higgins, D. G. (1997). The
CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by
quality analysis tools.Nucleic Acids Research,25, 4876–4882.
Wagner-Döbler, I., Beil, W., Lang, S., Meiners, M., & Laatsch, H. (2002). Integrated approach to
explore the potential of marine microorganisms for the production of bioactive metabolites.
Advances in Biochemical Engineering/Biotechnology,74, 207–238.
Xu, J. (2015). Bioactive natural products derived from mangrove-associated microbes.RSC
Advances,5, 841–892.