ORIGINAL RESEARCH
published: 31 January 2018
doi: 10.3389/fmicb.2018.00065
Frontiers in Microbiology | www.frontiersin.org 1 January 2018 | Volume 9 | Article 65
Edited by:
Iftikhar Ahmed,
Institute of Microbial Culture Collection
of Pakistan, National Agricultural
Research Centre, Pakistan
Reviewed by:
Samina Mehnaz,
Forman Christian College, Pakistan
Yuki Chan,
University of Hong Kong, Hong Kong
Kauser Abdulla Malik,
Forman Christian College, Pakistan
*Correspondence:
Fehmida Bibi
fehmeedaimran@yahoo.com
Specialty section:
This article was submitted to
Extreme Microbiology,
a section of the journal
Frontiers in Microbiology
Received: 16 November 2017
Accepted: 11 January 2018
Published: 31 January 2018
Citation:
Bibi F, Strobel GA, Naseer MI, Yasir M,
Khalaf Al-Ghamdi AA and Azhar EI
(2018) Microbial Flora Associated with
the Halophyte–Salsola imbricate and
Its Biotechnical Potential.
Front. Microbiol. 9:65.
doi: 10.3389/fmicb.2018.00065
Microbial Flora Associated with the
Halophyte–Salsola imbricate and Its
Biotechnical Potential
Fehmida Bibi 1*, Gary A. Strobel 2, Muhammad I. Naseer 3, Muhammad Yasir 1,
Ahmed A. Khalaf Al-Ghamdi 4 and Esam I. Azhar 1,4
1 Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia,
2Department of plant sciences, Montana State University, Bozeman, MT, United States, 3Center of Excellence in Genomic
Medicine Research, King Abdulaziz University, Jeddah, Saudi Arabia, 4Department of Medical Laboratory Technology,
Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia
Halophytes are associated with the intertidal forest ecosystem of Saudi Arabia and
seemingly have an immense potential for yielding useful and important natural products.
In this study we have aimed to isolate and characterize the endophytic and rhizospheric
bacterial communities from the halophyte, Salsola imbricata, In addition these bacterial
strains were identified and selected strains were further studied for bioactive secondary
metabolites. At least 168 rhizspheric and endophytic bacteria were isolated and of
these 22 were active antagonists against the oomycetous fungal plant pathogens,
Phytophthora capsici and Pythium ultimum. Active cultures were mainly identified with
molecular techniques (16S r DNA) and this revealed 95.7–100% sequence similarities
with relevant type strains. These microorgansims were grouped into four major classes:
Actinobacteria, Firmicutes, β-Proteobacteria, and γ -Proteobacteria. Production of fungal
cell wall lytic enzymes was detectedmostly in members of Actinobacteria and Firmicutes.
PCR screening for type I polyketide synthases (PKS-I), type II polyketide synthases
(PKS-II) and nonribosomal peptide synthetases (NRPS) revealed 13 of the 22 strains
(59%) were positive for at least one of these important biosynthetic genes that
are known to be involved in the synthesis of important antibiotics. Four bacterial
strains of Actinobacteria with potential antagonistic activity including two rhizobacteria,
EA52 (Nocardiopsis sp.), EA58 (Pseudonocardia sp.) and two endophytic bacteria
Streptomyces sp. (EA65) and Streptomyces sp. (EA67) were selected for secondary
metabolite analyses using LC-MS. As a result, the presence of different bioactive
compounds in the culture extracts was detected some of which are already reported for
their diverse biological activities including antibiotics such as Sulfamethoxypyridazine,
Sulfamerazine, and Dimetridazole. In conclusion, this study provides an insight into
antagonistic bacterial population especially the Actinobacteria from S. imbricata,
producing antifungal metabolites of medical significance and characterized taxonomically
in future.
Keywords: halophyte, antifungal activity, enzyme production, 16S rRNA gene sequence, LC/MS analyses
Bibi et al. Halophytic Plant Microbes
INTRODUCTION
Coastal dune plants consist of shrubs that are associated
with tropical and subtropical coastal areas. This ecosystem is
considered as a low nutrient environment due to the sparseness
of nitrogen and phosphorus (Frosini et al., 2012). Coastal dune
areas of the world usually represent a unique flora as a result of
variations in salinity, anaerobicity, and temperature fluctuations
during different seasons. All of these conditions make this habitat
unusual and thusmay have been selective for the presence of truly
unique microorganisms having a rich biodiversity. Under these
conditions microorganisms associated with these costal dune
plants play a role in their survival and perform diverse biological
functions in plants (Del Vecchio et al., 2015).
Bacteria associated with Coastal dune plants are responsible
for various activities including resistance against different
pathogens by producing different bioactive metabolites and plant
growth promotion compounds. Bacteria are associated with
these plants either as rhizospheric or endophytic forms and use
nutrients from plants for their growth (de Melo Pereira et al.,
2012). Endophytic bacteria get access to internal parts of plants
by producing enzymes to hydrolyze cell wall of plants (Cho et al.,
2007). These hydrolytic enzymes are also involved in defense of
plants against various bacterial and fungal pathogens (Luo et al.,
2013).
Endophytes produce metabolites that possess antimicrobial
activities (Strobel, 2003). Due to their beneficial effects and
broad range, endophytes have been isolated from plants widely
ranging from the poles of the earth to the steaming tropics.
Plants provide these microorganisms, particularly endophytic
bacteria and fungi, a nutrient-rich environment for their growth
and development (Strobel et al., 2004; de Melo Pereira et al.,
2012). Endophytic Actinobacteria associated with plant roots
or leaves are a source of novel potential bioactive substances.
Actinobacteria produce various secondary metabolites including
antibiotics, antitumor, immunosuppressive, and enzymes
widely used in industrial, agriculture, and pharmaceutical
industry (Manivasagan et al., 2014). Recently, various
antimicrobial compounds and enzymes have been isolated
from marine Actinobacteria (Hong et al., 2009; He et al., 2012).
Secondary metabolite salinosporamide A, isolated from a marine
Actinobacteria is currently in clinical trials as a cancer treatment
(Feling et al., 2003). Several other bioactive compounds have
been isolated from different marine Actinobacteria (Fu et al.,
2012).
The Genus Salsola is comprised of different halophyte species
including Salsola imbricata that inhabit the coastal area of
the Thuwal region in Jeddah, Saudi Arabia. Recent studies
on S. imbricata reported the presence of triterpenesaponins
and a new isorhamnetin from the methanolic extract of
this plant (Osman et al., 2016). In a recent study from
Saudi Arabia, phytochemical analysis of S. imbricata showed
presence of bioactive biphenylpropanoids i.e., biphenylsalsonoid
A and B. These two bioactive compounds exhibited antioxidant
and antibacterial activity against human pathogenic bacteria
(Oueslati et al., 2017). Although the coastal dune environment
is an unexplored source of natural compounds with diverse
activities there has been little or no work on the bacterial flora
of this plant. Based on previous studies, it would appear that
certain microbial flora components may be present in this plant
that has biotechnical potential. Thus, this study was undertaken
to explore potential antifungal microflora of halophyte that
may produce products of medicinal, agricultural, and industrial
significance.
MATERIALS AND METHODS
Collection of Plant Samples and Isolation
of Rhizobacteria and Endophytic Bacteria
The halophyte, S. imbricata was collected from the coastal area
of Jeddah, Saudi Arabia (22◦ 15′ 54′′ North, 39◦ 6′ 44′′ East).
We used soil, roots and leaves of plant sample for the isolation
of both rhizopheric and endophytic bacteria and the sample
plant were placed in sterile plastic bag at the time of collection.
For isolation of bacteria from soil associated with roots they
were dipped in sterile distilled water and serial dilutions were
made (10−3, 10−4, and 10−5) in filtered autoclaved sea water
(FAS) and spread in triplicate on four different media used
for culturing of bacteria. We have used half strength R2A (½
R2A) [0.25 g yeast extract, 0.25 g proteose peptone No. 3 (Difco),
0.25 g casamino acid, 0.25 g dextrose, 0.25 g soluble starch, 0.15 g
sodium pyruvate, 0.15 g K2HPO4, 0.03 g MgSO4], half Tryptic
soy agar (½ TSA) [Pancreatic Digest of Casein, 7.5g Papain Digest
of Soybean, 2.5 g Sodium Chloride, 2.5 g Agar, 15.0 g], marine
agar (MA) [Peptone, 5.0 g Yeast Extract, 1.0 g Ferric Citrate, 0.1 g
Sodium Chloride, 19.45 g Magnesium Chloride, 8.8 g Sodium
Sulfate, 3.24 g Calcium Chloride, 1.8 g Potassium Chloride,
0.55 g Sodium Bicarbonate, 0.16 g Potassium Bromide, 0.08 g
StrontiumChloride, 34.0mg Boric Acid, 22.0mg Sodium Silicate,
4.0mg Sodium Fluoride, 2.4mg Ammonium Nitrate, 1.6mg
Disodium Phosphate, 8.0mg Agar,15.0 g] and half nutrient agar
(½ NA) [Beef Extract, 1.5 g Peptone, 2.5 g Agar, 15.0 g] (Difco
Laboratories, Detroit, MI) in 1000ml filtered Sea water for
bacterial culturing. For isolation of endophytic bacteria, roots,
and leaves were surface sterilized. Both roots and leaves were
washed several times with tap water to remove adhering soil
particles and then sterilized by washing with disinfectants as
described previously (Bibi et al., 2012). After sterilization, small
pieces of sterilized root and leaf segments were ground in FAS
using a sterile mortar with pestle. Aliquots were further serially
diluted and plated in triplicate on four different mediamentioned
above. To suppress fungal growth, 50µg/ml cycloheximide was
mixed to the medium before pouring. The plates were incubated
at 28◦C for 2 weeks for bacterial growth.
Isolation of Rhizopheric and Endophytic
Actinobacteria
For the isolation of rhizosphere and endophytic Actinobacteria,
the same dilutions from soil, roots, and leaves were inoculated
onto starch-casein agar (Himedia) in sea water supplemented
with cycloheximide and nystatin (50µg/ml). Further, plates
were incubated at 28◦C for up to 10-12 days. By checking
morphological characteristics, Actinobacteria were selected and
Frontiers in Microbiology | www.frontiersin.org 2 January 2018 | Volume 9 | Article 65
Bibi et al. Halophytic Plant Microbes
subcultured using the same isolation medium. Individual
colonies were streaked to check purity of the strains and stored
in 15% (v/v) glycerol. All bacterial strains were labeled according
to acquisition numbers of the King Abdulaziz University special
infectious agents unit and stored at−70C.
Screening of Bacteria for Antifungal
Activity
All isolated bacteria were screened for their antifungal activity.
We used five different fungal pathogens i.e., Phytophthora
capsici (P. capsici), Pythium ultimum (Py. ultimum), (obtained
in this laboratory from Capsicum annuum and Cucumis sativus
respectively), Magnaporthe grisea (KACC 40415), Altenaria malli
(KCTC 6972) and Fusarium moniliforme (KCTC 6149) were
obtained from Korean Agricultural Culture Collection (KACC)
and Korean type culture collection center (KCTC). Antifungal
activity was determined by using the cross streak method
(Bibi et al., 2012). All isolates were streaked on PDA medium
supplemented with ½ R2A in sea water. Each 6mmmycelial disc
of freshly grown fungal pathogen was placed in the center of plate
perpendicular to streak of isolates at 4 cm distance from edges of
plate and incubated for 4–6 days at 28◦C. Antagonistic strains
were checked in duplicate for antagonistic activity evaluated by
measuring the inhibition zone of fungal pathogens around each
bacterial streak.
Bacterial DNA Extraction and 16S rRNA
Gene Sequencing
Genomic DNA was extracted from the selected antagonistic
bacterial isolates using a DNA extraction kit (Thermo Scientific,
Waltham, USA). To identify antagonistic bacteria, 16S rRNA
gene sequencing was performed. Approximately 1500 bp
fragment of the 16S rRNA gene was amplified using bacterial
universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′)
and 1492R (5′- GGTTACCTTGTTACGACTT-3′), the 16S
rRNA gene fragment was amplified under the following PCR
conditions: one cycle of 95◦C for 5min followed by 28 cycles of
95◦C for 1min, and annealing at 58◦C for 50 s with extension
at 72◦C for 50s, and a final extension step at 72◦C for
10min (Bibi et al., 2012). PCR products were purified using
PCR purification kit (Thermo Scientific, Waltham, USA), and
sequenced commercially (Macrogen, South Korea). Antagonistic
bacteria were identified by blast searches of their 16S rRNA
gene sequences obtained using the EzTaxon server (https://
www.ezbiocloud.net) (Kim et al., 2012) to identify antagonistic
bacteria. Phylogenetic positions of the antagonistic bacteria were
confirmed using CLUSTALX (Thompson et al., 1997) multiple
alignments of the bacterial sequences and BioEdit software (Hall,
1999) was used to edit the gaps. The neighbor-joining method
in the MEGA6 Programme with bootstrap values based on 1,000
replications was used for construction of the phylogenetic tree
based on the 16S rRNA gene sequences (Tamura et al., 2013).
Evaluation of Hydrolytic Enzymatic Activity
Antagonistic bacterial isolates were further evaluated for their
lytic enzyme production. Protease activity was checked using
skim milk ½ R2A agar plates. Bacteria exhibiting protease
production made clear zone on skim milk agar plates. Amylase
production was checked on starch medium. Starch hydrolysis
was seen as a clear zone on agar plates by positive strains
(Kumar et al., 2012). For lipase activity, tributyrin ½ R2A agar
medium was used. After 48 h of incubation at 28◦C a clear
zone was detected around bacterial colonies after hydrolysis
of tributyrin. To check cellulase activity, CMC agar (carboxy
methyl cellulose agar) medium was used. Bacteria were streaked
on plates with the respective enzyme medium and incubated
at 28◦C for 2 days. Congo red (0.1%) solution was added to
plates and put on orbital shaker for 30min and then rinsed
with 1MNaCl (Hendricks et al., 1995). Cellulase activity was
observed as a clear zone on CMC agar plates around bacterial
colonies.
Detection of Antimicrobial Genes
It has been well established that many antimicrobial products
contain polyketides. Thus, to determine if the antagonistic
bacteria might contain such enzymes, a search of the bacterial
genome was done to check for the presence of genes coding
for these enzymes. To detect biosynthetic genes, polyketide
synthetase I (PKS-I) and nonribosomal peptide synthetase
(NRPS) genes, the following primer pairs K1F/M6R (5′-
TSAAGTCSAACATCGGBCA-3′; 5′-CGCAGGTTSCSGTAC
CAGTA-3′) and A3F/A7R (5′GCSTACSYSATSTACACSTCS
GG-3′; 5′-SASGTCVCCSGTSCGGTAS-3′), respectively were
used (Ayuso-Sacido and Genilloud, 2005). Amplification of
polyketide synthetase II (PKS-II) gene was performed using
primer pair KSα/Kβ (5′-TSGCSTGCTTGGAYGCSATC-3′;
5′-TGGAANCCGCCGAABCCTCT-3′) (Metsa-Ketela et al.,
1999). PCR reaction mixture (25 µl) contained 12.5 µl of PCR
mastermix (promega), 1 µl of each primer, 1 µl of template
DNA, and 9.5 µl of Nuclease-Free Water. Amplifications were
performed under following PCR conditions: one cycle of 95◦C
for 5min followed by 28 cycles of 95◦C for 1min, and annealing
at 58◦C (K1F/M6R), 56◦C (A3F/A7R) and 62◦C (KSα/Kβ) for
50 s with extension at 72◦C for 50 s, and a final extension step at
72◦C for 10min. PCR amplification products were then checked
by using 0.8% agarose through gel electrophoresis, and bands of
1.2–1.4 kb, 600 bp, and 700–800 bp were detected as products of
PKS-I, PKS-II, and NRPS genes, respectively.
Optimization of Bacterial Culture
Conditions for Production Bioactive
Compounds
To optimize culture conditions of selected bacterial strains
four different media i.e., ½ R2A broth, ½ TSB, ½NB in sea
water and Marine broth in distilled were used for culturing.
Bacterial cultures were grown for 72 h and after every 24 h
optical density (OD) was checked and antifungal activity was
assessed against P. capsici, Py. ultimum using the disc diffusion
method. The effect of temperature was optimized within the
range of temperatures (20–40◦C) in ½ R2A broth. For pH
optimization, different ranges of pH values (5–12) were used
for the growth and antifungal compound production in ½ R2A
broth.
Frontiers in Microbiology | www.frontiersin.org 3 January 2018 | Volume 9 | Article 65
Bibi et al. Halophytic Plant Microbes
LC-MS Analysis of Bacterial Culture
In order to assess the actual presence of antimicrobial products in
bacterial cultures that had been previously screened and shown
to be biologically active we performed LC-MS analyses of the
bacterial supernatants. A Bacterial culture (5ml) was placed on
−80◦C for 5min, and then transfer to 37◦C water bath for 5min
and this procedure was repeated 5 times. It was centrifuged
at 15,000 g for 10min and transfer 3ml supernatant fluid was
transferred to a tube. To it was added 12ml of acetonitrile and
vortexed for 30 s. It was centrifuged again at 15,000 g for 10min
and 300 µl supernatant was taken for LC-MS metabolomic
analysis. The injection volume was 3 µl and samples were
analyzed on an Agilent 6540B TOF/Q-TOF Mass Spectrometer
coupled with an Agilent 1290 UPLC andDual AJS ESI ion source.
An ACQUITY UPLC HSS T3 (100 × 2.1mm, 1.8 um) column
and pre-column (Phenomenex Security Guard
TM
) was used to
separate the sample. The column temperature was set to 45◦C
and flow rate was 0.5 ml/min. The acquisition range was from
50 m/z to 1500 m/z and the scan rate was 1.00 spec/sec. MS
conditions were set as follow: capillary voltage 3500V, nebulizer
pressure 35 psi, drying gas 10 L/min, gas temperature 325◦C,
vaporizer 200V, voltage charge 1000V; negative-ion mode
capillary voltage 3500V, corona negative 15.0V, fragmentor
175V, skimmer1 65.0V, octopole RF Peak 750V; positive ion
mode capillary voltage 3500V, corona positive 4.0 V, fragmentor
175V, skimmer1 65.0V, and octopole RF Peak 750V. Raw data
was imported to the Agilent MassHunter Qualitative Analysis
B.06.00 software. Metabolites were identified by using National
Institute of Standards and Technology database (NIST) database.
Further bioactive metabolites from other secondary metabolites
in culture extract were identified using different public
databases such as PubChem (http://pubchem.ncbi.nlm.nih.gov/),
ChemSpider (http://www.chemspider.com/), SciFinder (http://
www.cas.org/products/scifinder), and ChEMBL (https://www.
ebi.ac.uk/chembl/).
Nucleotide Sequence Numbers
Nucleotide sequences of the antagonistic bacteria isolated from
the halophyte have been deposited in the GenBank database
under accession numbers KY436424–KY436445.
RESULTS
Isolation of Rhizospheric and Endophytic
Bacteria
In this study, rhizospheric and endophytic bacteria were isolated
from the halophyte, S. imbricata growing in a selected site on
the Saudi Arabian peninsula. A total of 168 rhizospheric and
endophytic bacteria were isolated from soil, roots, and leaves
samples. Various methods were used to acquire these bacteria
including the use of four different culturing media. It turns out
that both high and low nutrient medium content favored the
growth of different groups of bacteria. It has been noted that
the ½ R2A in SW is a favorable medium for the isolation of
bacteria from the marine environment. Thus, all isolated bacteria
culture on different media were sub-cultured on ½ R2A in
SW. In total 168 bacteria were isolated from different parts of
halophyte and soil as well (Table 1). From soil, 85 rhizobacteria
were isolated by culturing on different media listed above for
isolation. Roots samples used for isolation of endophytic bacteria
yielded 48 endophytic bacteria. While from leaves 35 bacteria
were recovered. Details of bacteria number their antagonistic
activity and dominance of different bacterial phyla is mentioned
in Table 1.
Screening of Bacteria against Pathogenic
Fungi
All isolated bacteria were tested for their antagonism against
plant pathogenic oomycetes. Of the 168 bacteria isolated, 22
(13%) displayed inhibition to both oomycete plant pathogens
that was the established bioassay test. The percentage of
antagonistic bacteria varied in different parts of halophyte, being
highest in roots (n= 9; 5.3%), followed by soil (n= 8; 4.7%), and
leaves (n = 5; 2.9%). These antagonistic bacterial isolates were
tested further against three other fungal pathogens. Different
groups of antagonistic bacteria were identified from S. imbricata,
and Actinobacteria was the dominant phylum (Table 1. Both
rhizospheric and endophytic antagonistic Actinobacteria i.e.,
Nocardiopsis dassonvillei (EA52), Pseudonocardia sp. (EA58),
Arthrobacter sp. (EA59), and Streptomyces sp. (EA64) showed
strong inhibition against the oomycetes (Table 2). Some of
the antagonistic isolates (EA56, EA62, EA68-EA70) showed
inhibition against oomycetes but did not show inhibition against
other pathogenic fungi used (Table 2).
Identification and Phylogenetic Analysis of
Rhizopheric and Endophytic Antagonistic
Bacteria
In total, 22 antagonistic bacteria were identified by using 16S
rRNA gene sequence analysis. Result of sequencing showed that
eight different genera of bacteria were identified belonging to four
major classes: Actinobacteria (n = 15; 68%), Firmicutes (n = 5;
23%), γ -Proteobacteria (n = 1; 4.5%), and β-Proteobacteria
(n = 1; 4.5%). Sequence identities of antagonistic bacteria
were recorded from 95.7−100% (Table 1). Sequencing results
indicated Actinobacteria as the dominant phylum and 26% of
TABLE 1 | Distribution of rhizopheric and endophytic antagonistic bacteria
isolated from halophyte.
Isolation sourcea Number of
isolatesb
Number of
antagonistc
Antagonists
(%)d
Dominant
phylume
Rhizopheric (Soil) 85 8 4.7 Actinobacteria
Endophytes (Roots) 48 9 5.4 Actinobacteria
Endophytes (Leaves) 35 5 2.9 Firmicutes
168 22 13% Actinobacteria
a Isolation source of rhizopheric and endophytic bacteria.
bTotal number of rhizopheric and endophytic bacteria isolated from soil, roots and leaves
of halophyte.
cTotal number of antagonistic rhizopheric and endophytic bacteria.
dPercentage of antagonistic rhizopheric and endophytic bacteria.
eDominant phylum in all rhizopheric and endophytic antagonistic bacteria.
Frontiers in Microbiology | www.frontiersin.org 4 January 2018 | Volume 9 | Article 65
Bibi et al. Halophytic Plant Microbes
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Frontiers in Microbiology | www.frontiersin.org 5 January 2018 | Volume 9 | Article 65
Bibi et al. Halophytic Plant Microbes
the antagonistic isolates belonged to the genus Streptomyces
(Table 1). Using 16S rRNA gene data, a neighbor Joining (NJ)
phylogenetic tree was constructed (Figure 1). High bootstrap
values were recovered in phylogenetic tree. A separate cluster has
been generated for isolates of class Actinobacteria. Antagonistic
strains in this cluster were identified with high bootstraps
values (51–100%). Representatives of this class belong to four
different genera i.e., Nocardioides, Nocardiopsis, Arthrobacter,
and Streptomyces. One strain of β-Proteobacteria was identified
with separate cluster also showing high bootstrap value (100%).
One strain of γ -Proteobacteria made a distinct cluster with
closely related type strain Pseudomonas sp. (EA54) with high a
bootstrap value of 100 %. Antagonistic bacterial strains of class
Firmicutes were placed in separate cluster belonging to the genus
Bacillus. In this study, two antagonistic endophytic bacterial
strains were also identified showing low 16S rRNA gene sequence
similarity (<98%). One strain of Actinobacteria i.e., Streptomyces
sp. (EA64) and one strain belonging to Firmicutes i.e., Bacillus
sp. (EA68) were identified with similarities of <98% to the
closest type strains (Table 3). Four antagonistic Actinobacteria
were selected for secondary metabolites identification depending
on their antagonistic activity against oomycetes.
Enzymatic Activities of Antagonistic
Bacteria
Antagonistic bacteria were evaluated for their ability to produce
fungal cell wall lytic enzymes. Protease, amylase, lipase, and
cellulase activities of these bacteria were examined (Table 2).
The number of bacteria exhibiting protease activity (n = 9;
41%) was high as compared to other enzymatic activities.
Most of protease producing bacteria were endophytes (n = 6)
and recovered from leaves and roots of halophyte. Amylase
activity was observed for 7 (32%) antagonistic bacteria. Mostly
strains of Actinobacteria showed amylase activity. Endophytic
strains of Actinobacteria exhibited high production of amylase.
Lipase activity was observed in 7 (32%) antagonistic bacteria.
Strains of Actinobacteria and Firmicutes showed high production
of protease. Cellulase activity was observed in only 1 (4.5%)
bacterial strain. A strain of N. dassonvillei (EA52) exhibited
cellulase activity belonged to Actinobacteria.
Detection of PKS and NRPS Genes
Bacteria possess NRPS/PKS gene clusters especially common
in the phyla Proteobacteria, Actinobacteria and Firmicutes.
Antagonistic bacterial isolates from this study showed that of
FIGURE 1 | Phylogenetic distribution of antagonistic bacteria isolated from S.imbricata 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 inferred from the 16S rRNA gene sequences (1 kb) by using the
neighbor-joining method from distances computed with the Jukes-Cantor algorithm. Bootstrap values (1,000 replicates) are shown next to the branches. GenBank
accession numbers for each sequence are shown in parentheses. Bar, 0.01 accumulated changes per nucleotide. Isolates selected for bioactive metabolites
identification are highlighted.
Frontiers in Microbiology | www.frontiersin.org 6 January 2018 | Volume 9 | Article 65
Bibi et al. Halophytic Plant Microbes
TABLE 3 | Tentative novel taxa based on partial 16S rRNA gene sequence analyses from this study.
Source Similarity Identity% Family Gram stain
Rhizosphere (Soil) Bordetella avium ATCC 35086 T 98.9 Alcaligenaceae Gram-negative
Rhizosphere (Soil) Nocardioides deserti SC8A-24 T 98.9 Nocardioidaceae Gram-positive
Endophytic (Root) Streptomyces phaeopurpureus NRRL B-2260 T 98.8 Streptomycetaceae Gram-positive
Endophytic (Root) Streptomyces diastatochromogenes ATCC 12309T 97.3 Streptomycetaceae Gram-positive
Endophytic (Root) Bacillus sonorensis NBRC 101234 T 95.7 Bacillaceae Gram-positive
Endophytic (Leaf) Streptomyces europaeiscabiei KACC 20186T 98.2 Streptomycetaceae Gram-positive
Endophytic (Leaf) Bacillus halosaccharovorans E33T 98.9 Bacillaceae Gram-positive
22 isolates, PCR amplification revealed that 13 strains (59%)
were positive for at least one of the biosynthetic genes checked
(Table 2). For the NRPS gene, 8 (36%) strains were positive.
NRPS genes were detected in both classes of Actinobacteria
and Firmicutes. The PKS-I gene was detected in only 4 (18%)
antagonistic bacterial strains. While PKS-II gene was detected
in 7 (32%) strains. All three genes PKS-I and PKS-II and
NRPS were detected in strain Bacillus sp. (EA72). Three
isolates belong to Actinobacteria, Streptomyces enissocaesilis
sp. (EA52), Streptomyces sp. (EA63), and Streptomyces sp.
(EA64) were positive for the presence of both NRPS and
PKS-II.
Identification of Strain EA52, EA58
(Rhizobacteria), and EA65, EA67
(Endobacteria) Metabolites by LC-MS
On the basis of high inhibition activity against fungal pathogens
we have selected four antagonistic bacterial strains from our
study to identify secondary metabolites. These four antagonistic
isolates belong to class Actinobacteria i.e., Nocardiopsis sp.
(EA52) and Pseudonocardia sp. (EA58) were rhizobacteria
while Streptomyces sp. (EA65) and Streptomyces sp. (EA67)
are endophytes. Production of antifungal compounds was
influenced by culturing in R2A as culture medium using
optimum culture conditions. The maximum antifungal activity
was observed after 48 h of growth at 28◦C with pH 7.5.
Metabolites in cultures extract of selected bacteria were analyzed
using the LC-MS. LC-MS technique confirms presence of
various bioactive compounds although not novel but already
known for their bioactivity. Identification of metabolites
was determined by LC-MS analysis and comparing results
from NIST database. LC-MS analysis of culture extract of
these selected strains identified different bioactive metabolites
(Table 4). Strain of Nocardiopsis sp. (EA52) produced 7
identified peaks of different bioactive metabolites in both
the positive- and negative-ion mode (Figures 2A,B). These
compounds include Thiabendazole, Sulfamethoxypyridazine, N-
Nitrosodiethylamine, Ibuprofen, Laberalol, Isoxsuprine, and
Oxibendazole. For Pseudonocardia carboxydivorans (EA58), 2
bioactive compounds have been detected among hundreds
of peaks known for other different metabolites. These three
compounds include Sulfamethoxypyridazine and Dimetridazole
(Figures 2C,D). Strain EA65 genetically closely associated as
a Streptomyces diastatochromogenes showed presence of nine
different bioactive compounds in the culture extract i.e.,
Sulfamethoxypyridazine, Dimetridazole, Salbutero, Moxidectin,
Atenolol, Timolol, Benzydamine, Acebutolol, and Bambuterol
(Figures 2E,F). These bioactive secondary metabolites are
already known for their biological activities. LC-MS analysis of
strain EA67, Streptomyces europaeiscabiei showed the presence
of six bioactive compounds among several compounds detected.
These bioactive compounds include, Sulfamethoxypyridazine,
Benzamide, Sulfamerazine, Dimetridazole, Metronidazole-oh,
and Carazolol (Figures 2G,H). These compounds are already
known for antimicrobial, antiphytopathogenic and biocontrol
properties.
DISCUSSION
There is a need for the discovery of new drugs due to the
emergence of resistant bacteria to different antibiotics and cancer
cells to anticancer drugs. Currently, marine habitats have been
identified as a source of discovering many important novel
therapeutic agents (Malve, 2016). S.imbricata, is a halophyte
containing various chemical compounds of medicinal use and it
is widely spread in the sea shore areas in Saudi Arabia. Generally
halophytes with traditional health benefits in folk medicine are
a source of attraction for modern comprehensive microbial
and chemical investigations. In our previous study (Bibi et al.,
2017) we have isolated two rhizobacteria from Salsola exhibiting
cellulase and amylase activity on culturing media. One strain
EA151 with cellulase activity belonged to Firmicutes while other
strain EA152 exhibited amylase activity closely related to type
strain of Actinobacteria.
In this study, rhizopheric and endophytic bacterial
populations with potent antagonistic activity against
phytopathogens were studied. One hundred and sixty eight
rhizopheric and endophytic bacteria were isolated and only 22
were found to be potent antagonists of oomycetes. Different
cultivation media were used to culture bacteria to isolate novel
isolates with unique bioactivities. A study has reported that
high nutrient medium favors growth of diverse groups of
bacteria, while low nutrient media increase number of bacteria
(Vishnivetskaya et al., 2000). To our knowledge, this work is
the first study on the isolation, screening, characterization and
metabolites identification of both rhizospheric and endophytic
bacteria from S. imbricata. In this study, culture-dependent
approach was used to study the diversity of antagonistic bacteria
Frontiers in Microbiology | www.frontiersin.org 7 January 2018 | Volume 9 | Article 65
Bibi et al. Halophytic Plant Microbes
TABLE 4 | Secondary metabolites found in crude extract of selected four Actinobacteria strains.
No Name Formula RT m/z MASS Score Diff(DB, ppm) Mode Height Area
STRAIN EA52
1 Thiabendazole C10 H7 N3S 0.962 201.037 201.0366 44.89 −2.4 Negative 36878 184023
2 Sulfamethoxypyridazine C11 H12 N4 O3S 1.125 279.0582 280.0655 75.22 −9.01 Negative 44633 255086
3 N-Nitrosodiethylamine C4 H10 N2 O 1.579 215.0641 102.0784 46.98 8.48 Negative 23538 232204
4 Ibuprofen C13 H18 O2 2.042 189.127 206.1303 92.03 2.07 Positive 62696 509785
5 Laberalol C19 H24 N2 O3 2.8 329.1839 328.1766 81.9 6.46 Positive 54189 463656
6 Isoxsuprine C18 H23N O3 2.892 302.1733 301.1658 82.96 6.73 Positive 139154 2034111
7 Oxibendazole C12 H15 N3 O3 3.533 250.1187 249.1114 97.67 −0.32 Positive 126155 961667
STRAIN EA58
8 Sulfamethoxypyridazine C11 H12 N4 O3S 1.117 279.0575 280.0647 67.12 −6.21 Negative 15321 77965
9 Sulfadiazin C10 H10 N4 O2S 2.683 249.0475 250.055 72.64 −10.1 Negative 29954 210781
10 Dimetridazole C5 H7 N3 O2 4.045 159.0876 141.0527 70.14 8.14 Positive 111793 948315
STRAIN EA65
11 Sulfamethoxypyridazine C11 H12 N4 O3S 1.126 279.0574 280.0649 80.47 −6.78 Negative 47164 293920
12 Sulfadiazin C10 H10 N4 O2S 2.686 249.0474 250.0548 74.78 −9.52 Negative 41010 344049
13 Sulfacetamide C8 H10 N2 O3S 1.793 215.0474 214.0401 72.66 5.06 Positive 68031 563882
14 Dimetridazole C5 H7 N3 O2 4.039 159.0881 141.0535 82.65 2.25 Positive 211556 1889105
15 Salbuterol C13 H21N O3 15.786 239.1764 239.1532 47.28 −4.28 Positive 1181230 8578877
16 Moxidectin C37 H53N O8 16.671 662.3704 639.3739 49.37 4.97 Positive 95729 1314871
17 Atenolol C14 H22 N2 O3 17.318 267.1729 266.1657 77.62 −10.11 Positive 907768 7927165
18 Timolol C13 H24 N4 O3S 21.415 317.1641 316.1571 73.26 −0.54 Positive 67063 538273
19 Benzydamine C19 H23 N3 O 21.419 310.1884 309.1797 21.59 14.35 Positive 89361 929352
20 Acebutolol C18 H28 N2 O4 21.711 336.2052 336.2045 59.81 1.09 Positive 88941 887397
21 Bambuterol C18 H29 N3 O5 21.714 367.2376 367.2098 51.49 2.53 Positive 58314 570222
STRAIN EA67
22 Sulfamethoxypyridazine C11 H12 N4 O3S 1.122 279.0576 280.0651 80.42 −7.33 Negative 30594 156782
23 Sulfadiazin C10 H10 N4 O2S 2.682 249.0471 250.0545 78.19 −8.26 Negative 44098 367641
24 Sulfanilamide C6 H8 N2 O2S 0.748 173.0376 172.0307 91.12 −0.47 Positive 95732 1075748
25 Sulfadiazin C10 H10 N4 O2S 1.223 251.058 250.0508 65.17 6.67 Positive 99002 476334
26 Sulfacetamide C8 H10 N2 O3S 1.8 215.0475 214.0404 90.98 3.98 Positive 142535 1446212
27 Benzamide C7 H7N O 2.72 103.0426 121.0525 37.43 1.82 Positive 641812 5370210
28 Sulfamerazine C11 H12 N4 O2S 2.991 265.0734 264.0662 65 7.1 Positive 59659 389438
29 Dimetridazole C5 H7 N3 O2 4.059 159.0879 141.0531 75.55 5.01 Positive 149866 1286924
30 Metronidazole–oh C6 H9 N3 O4 4.06 188.0669 187.0597 95.68 −1.89 Positive 1776794 17824964
31 Carazolol C18 H22 N2 O2 20.924 303.1474 298.1688 42.24 −2.22 Positive 81193 668836
from halophyte against oomycetes plant pathogens. Screening of
168 rhizospheric and endophytic bacteria and their 16S rRNA
gene sequencing resulted in 22 different bacteria belong to four
major classes. Bioactive compounds production by rhizo and
endophytic bacteria is one of the phenomenon used by bacteria
to defend the host against different pathogens.
Antagonistic bacteria belong to four major classes, Firmicutes,
γ -Proteobacteria, and β-Proteobacteria. Our results show that
Actinobacteria is recognized as a dominant phylum among
these antagonitistic bacteria isolated from halophyte (Table 1).
Actinobacteria are distributed in various ecological habitats
especially in terrestrial and aquatic ecosystems where they play a
potential role in decomposition of plants and animals remains.
They are biotechnologically important due to their ability to
produce secondary metabolites (Berdy, 2005). Therefore, we
have selected four different strains of Actinobacteria for further
study of bioactive metabolites produced by these antagonistic
rhizopheric and endophytic bacteria. Results of 16S rRNA
sequence analyses showed a diversity of different genera of
Actinobacteria and the genus Streptomyces remain which are
dominant in our study. Results of this study are also compatible
with previous studies wheremany novelActinobacteria have been
recovered from different unexplored marine sources (Fiedler
et al., 2005; Bull and Stach, 2007). Halophytes growing near
the sea shore in tropical and subtropical areas have become
valuable sources for the isolation of microbial flora especially
in the group Actinobacteria because of the chemodiversity of
environmental factors prevailing there. The presence of major
numbers of Streptomyces strains amongst antagonists from the
halophyte in this study are in accordance with the results reported
Frontiers in Microbiology | www.frontiersin.org 8 January 2018 | Volume 9 | Article 65
Bibi et al. Halophytic Plant Microbes
FIGURE 2 | Spectra of LC/MS analysis showing detection of various bioactive
metabolites. Nocardiopsis sp. (EA52) (A) negative mode LC/MS analysis
(B) positive mode LC/MS analysis (C) negative mode LC/MS analysis of
Pseudonocardia sp. (EA58) (D) positive mode LC/MS analysis (E) negative
mode LC/MS analysis of Streptomyces sp. (EA65) (F) positive mode LC/MS
analysis (G) negative mode LC/MS analysis of Streptomyces sp. (EA67)
(H) positive mode LC/MS analysis.
previously (Eccleston et al., 2008; Ravikumar et al., 2012). In
Actinibacteria, the most active bacterial strains in addition to
Streptomyces are,Nocardiopsis, Pseudonocardia, andArthrobacter
which have been reported for their inhibitory activities and
production of antimicrobial metabolites (Anandan et al., 2016).
The metabolic diversity of Streptomycetes is remarkable
and novel strains with new bioactive compounds including
antibiotics have been isolated from extreme habitats (Fiedler
et al., 2005). Until now, several studies have reported diverse
groups of antimicrobial compounds by bacteria isolated from
halophytic endophytes, such as steroids, peptides, alkaloids,
terpenoids, quinines, flavonoids, and phenols (Newman and
Cragg, 2007). Our results showed that most of the endophytes
in this study belonging to Streptomyces and were recovered from
root samples. This finding is supported by previous results where
maximum number of endophytes were recovered from roots
specimens (Taechowisan et al., 2003; Passari et al., 2015) Thismay
be due to the reason that the rhizosphere has a high concentration
of nutrients and various bacteria enter different root tissues of
the host plant through the rhizosphere and become endophytic
in their mode of life (Rosenblueth and Martinez-Romero, 2006).
The second dominant class of bacteria in this study was
Firmicutes. The group of bacteria belonging to the class
Firmicutes are commonly found as they are easily culturable.
Also, from marine sources, species of Bacillus are dominant
for the production of antibacterial, antifungal antibiotics,
surfactants, and lytic enzymes (Mondol et al., 2013). Endophytic
bacterial strains of Bacillus from halophytes are already
reported to show inhibition against bacterial and fungal plant
pathogens (Menpara and Chanda, 2013). In this study, only two
antagonistic rhizopheric and endophytic bacteria were related
to the class Proteobacteria belonging to two different classes
γ -Proteobacteria and β-Proteobacteria. Members of class γ -
Protobacteria from marine source produce the highest number
of secondary metabolites (Long and Azam, 2001).
Hydrolytic enzymes from marine bacteria are produced and
used by them in catalyzing different biochemical processes
in the marine environment (Thatoi et al., 2013). In addition
to production of useful secondary metabolites and antibiotics
Actinobacteria from marine sources are also known for the
production of lytic enzymes of industrial use (Anandan et al.,
2016). In this study, mostly strains of Actinobacteria and
Firmicutes were active candidates for production of different
enzymes. Protease, lipase and amylase activity was mostly
observed (Table 2). Only one strain EA59 exhibited protease,
amylase and lipase activity. This strain is endophytic and
belongs to Actinobacteria. It seems as if the productions of
these hydrolytic enzymes is necessary for antagonism as well
as for intracellular colonization of bacteria in the host plant.
Only four strain of rhizobacteria belong to Actinobacteria
were able to produce at least one type of enzyme tested
in this study. While most of the endophytic bacteria were
able to show enzymatic activities belong to Actinobacteria.
Marine Actinobacteria have the potential to produce extracellular
enzymes of industrials use. These extracellular enzymes are
usually used by endophytic bacteria for their antagonism against
different pathogens invading host plants (Zhao et al., 2016).
Frontiers in Microbiology | www.frontiersin.org 9 January 2018 | Volume 9 | Article 65
Bibi et al. Halophytic Plant Microbes
Polyketide synthetase (PKS) and nonribosomal polyketide
synthetase (NRPS) genes are considered as hallmarks for the
production of secondary metabolites. These biosynthetic genes of
bacterial secondary metabolites are involved in the biosynthesis
of many bioactive products. The presence of the biosynthetic
genes PKS and NRPS in rhizospheric and endophytic bacteria
associated with this halophyte suggests that these beneficial
bacteria are dominant producers of bioactive natural products.
Amplification of these three biosynthetic gene clusters domains
is an indirect approach to test the knowledge that bioactive
products are being produced by the organism. Of 22 isolates
screened for detection of PKS-I, PKS-II, and NRPS gene, only
13 strains were positive for at least one of the biosynthetic genes
tested. Several species of Streptomyces possess two types of PKSs.
One is responsible for antibiotic synthesis, while the other is
involved in spore pigments production in various Streptomyces
species (Bergh and Uhlén, 1992). In this study, PKS-II and
NRPS were detected in most of the Streptomyces. Among them,
strains EA52, EA61, EA63, and EA64 were positive for the
presence of NRPS and PKS-II, and also exhibited antifungal
activity against tested pathogens (Table 2). While strains EA53,
EA56, EA59, EA60, EA62, EA68-EA71 were negative for the
presence of PKS-I, PKS-II, and NRPS genes and positive for
antifungal activity. Therefore, there is no correlation between
presence of PKS and NRPS gene and antimicrobial properties
(Qin et al., 2009). It is evident that NRPS PKS-I and PKS-II
gene screening using PCR in different bacterial strain help to find
out potential strain of bacteria producing important secondary
metabolites.
Strains of Streptomycetes are active producers of antimicrobial
compounds and more than 70% of antibiotics in the world
are produced by them. Endophytic Streptomycetes occupy a
biologically important niche in tissues of the host plant, taking
nutrition from host and in turn providing protection to the
plant. These endophytes produce important metabolites that
are not toxic to their host therefore, can be considered as
important bioactive metabolites in drug discovery (Castillo et al.,
2007). Selection of Actinobacteria for identification of active
secondary metabolites resulted in a number of known synthetic
bioactive compounds not of microbial origin (Table 4). Based
upon antifungal activity and low 16S rRNA gene similarity
four out of twenty-two rhizopheric and endophytic strains
were ultimately selected for LC-MS analyses. Production
of antifungal compounds was influenced by culturing the
organism in R2A as a culture medium using optimum culture
conditions. The maximum antifungal activity was observed after
48 h of growth with pH 7.5 at 28◦C. Metabolites in cultures
extracts of selected bacteria were analyzed using LC-MS. Two
rhizopheric strains namely EA52 closely related to Nocardiopsis
sp. and EA58 showed similarity with Pseudonocardia sp.
in producing diverse metabolites including Thiabendazole,
Sulfamethoxypyridazine, N-Nitrosodiethylamine, Ibuprofen,
Laberalol, Isoxsuprine, Oxibendazole, and Dimetridazole.
Strain EA52 showed close similarity to Nocardiopsis sp.
This marine non-Streptomycete genus of Actinobacteria,
contained a variety of bioactive compounds and showed a
wide range of activities including cytotoxicity, antibacterial,
antifungal, and anti-angiogenesis. Nocardiopsis sp. contained
α-pyrone (nocapyrone S1) and (4-aminophenyl) acetic acid,
N-(2- hydroxyphenyl)-acetamide, cyclo-(L-Pro-L-Val), cyclo-
(L-Pro-L-Leu), and cyclo-(L-Pro-L-Ile) when evaluated for
secondary metabolite production (Zou et al., 2017) but
different secondary metabolites have been detected in closely
related strain EA58 in the present study (Figures 2A,B).
Antimicrobial activity of strains of Pseudonocardia is already
evident from previous studies but no such antifungal activity
and metabolites detected in our study were produced from
the closely related strain EA58 (Figures 2C,D; Tanvir et al.,
2016).
Two endophytic Streptomycetes EA65 and EA67 were
analyzed by LC-MS and they exhibited spectra for 15 bioactive
compounds including antibacterial, antifungal, antiprotozoal and
anthelmintic compounds. Strain EA65 showed close similarity
at the 97% level to a rhizobacterium S. diastatochromogenes, a
promising source of antifungal metabolites and novel natural
antibiotics and used as biocontrol agents (Shentu et al., 2016).
S. diastatochromogenes reportedly produces polyketomycin,
momofulvenoneA, azdimycin, toyocamycin, concanamycin,
and oligomycins. Strain EA65 exhibited the presence of
bioactive compounds not reported in S. diastatochromogenes
including Sulfamethoxypyridazine, Dimetridazole, Salbutero,
Moxidectin, Atenolol, Timolol, Benzydamine, Acebutolol,
and Bambuterol (Figures 2E,F). Another endophytic strain
EA67 related to S. europaeiscabiei showed the presence
of bioactive compounds such as Sulfamethoxypyridazine,
Benzamide, Sulfamerazine, Dimetridazole, Metronidazole-oh,
and Carazolol (Figures 2G,H). These compounds are already
known for antimicrobial, antiphytopathogenic and biocontrol
properties. Sulfamethoxypyridazine, Nitroimidazole including
Dimetridazole and Metronidazole-oh and Moxidectin are
major antimicrobial compounds detected in all four strains
in this study. Sulfamethoxypyridazine was detected in all four
strains in our study. These antibacterial sulfonamides are
synthetic antimicrobial agents used as antibiotics in different
inflammatory diseases (Vicente and Pérez-Trallero, 2010).
This is first study showing the isolation and screening of
antagonistic bacteria from S. imbricata and the identification of
active metabolites from crude extracts. Compounds produced
by these strains although not new, have been not reported
in literature from other organisms and most of them are
synthetic so this is the first reported of these Actinobacteria
as a natural source for production of these antimicrobial
compounds.
In conclusions, present study of antagonistic bacteria from
S. imbricata exhibited wide range antifungal and enzymatic
activities indicating their industrial, biotechnological and
agricultural potential. Further analyses of bioactive metabolites
from selected Actinobacteria exhibited production of known
antibiotics and bioactive compounds of synthetic nature not
reported from bacteria before. These results suggest that
halophytes are potential source of bioactive metabolites via their
associated bacterial microflora. This study points to an enormous
potential of discovering novel bioactive compounds using the
methodology described. Currently we are working on some
Frontiers in Microbiology | www.frontiersin.org 10 January 2018 | Volume 9 | Article 65
Bibi et al. Halophytic Plant Microbes
selected novel strains for deep bioactive analysis and genome
sequencing that will be presented in our future work.
AUTHOR CONTRIBUTIONS
FB and GS: designed the study and write manuscript; MN and
EA: identified the plant samples and help in collection of sample
and experimental work; MY and AK collect and analyzed the
data. All authors read and approved the final manuscript.
ACKNOWLEDGMENTS
This project was funded by the National Plan for
Science, Technology and Innovation (MAARIFAH)–
King Abdulaziz City for Science and Technology-the
Kingdom of Saudi Arabia-award number (12-BIO2724-03).
The authors also, acknowledge with thanks Science and
Technology Unit, King Abdulaziz University for technical
support.
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Conflict of Interest Statement: The authors declare that the research was
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be construed as a potential conflict of interest.
Copyright © 2018 Bibi, Strobel, Naseer, Yasir, Khalaf Al-Ghamdi and Azhar. This
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