Indian Journal of Geo-Marine Sciences
Vol. 43(4), April 2014, pp. 571-579
Molecular phylogeny and plant growth promoting traits of endophytic
bacteria isolated from roots of seagrass Cymodocea serrulata
Polpass Arul Jose, Ilangovan Shanmuga sundari, Kunjukrishnan Kamalakshi Sivakala &
Solomon Robinson David Jebakumar*
Department of Molecular Microbiology, School of Biotechnology,
Madurai Kamaraj University, Madurai - 625 021, India
[E.mail: [email protected] ]
Received 5 April 2013; revised 19 September 2013
In this study, phylogeny and growth promoting attributes of bacterial endophytes isolated from root tissue of seagrass
were investigated. Sequence analysis of 16S rDNA among the isolates displayed the presence of bacterial members affiliated
to six different genera: Vibrio, Photobacterium, Bacillus, Aerococcus, Saccharomonospora and Kocuria. Most of the isolates
shared high level 16S rDNA sequence similarity with bacteria previously reported from marine water column, crustaceans,
corals and terrestrial rhizosphere. Plant growth promoting (PGP) attributes including ability to fix nitrogen, solubilize
phosphate and produce ammonia, acetoin and indolic compound were assessed in all the isolates. Genetically diverse,
endophytic isolates with different PGP abilities showed close relatedness with bacterial inhabitants of marine ecosystem
and likely to have ecological relevance. It implies the need of further studies to validate the relationship between the isolated
endophytes and seagrass for sustenance of marine ecology.
[Keywords: Endophytes, Cymodocea serrulata, Seagrass, Phylogeney, PGP traits ]
Introduction
Diverse communities of endophytic bacteria are
in association with roots of terrestrial and aquatic
plants, without exerting any adverse effects to the
host1. The endophytic bacterial population comprises
both Gram-positive and Gram-negative bacteria 2;
often favour plant growth either by ensuring the
availability of limited nutrients or responding to
pathogens, stress and pollutants through mutualistic
bacteria-plant interactions3-7. There have been several
reports on composition and ecological roles of
symbiotic bacterial communities associated with
terrestrial and fresh water plants; however, the
presence and relevance of endophytic bacterial
communities in marine plants remain almost
unexplored8.
* Corresponding author:
Seagrasses are primary producers, developing
lush and highly productive meadows in shallow
coastal environments with several ecological and
biogeochemical significances9-11. Seagrasses stabilize
water quality by filtering suspended matter in the
water column12, prevent surface fouling by inhibiting
marine pathogens13,14 and provide a link between
sediment and water column nutrient cycles15-16. These
ecological roles of seagrasses are significantly
contributed by the rich associated bacterial
communities17-19,14. Microbial activities in seagrass
bed sediments showed strong seasonality and were
highest when the plants were actively growing20,21 and
suggest mutual interactions between the seagrasses
and associated microbial communities.
Earlier studies on bacterial communities
associated with seagrasses have been focused on
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INDIAN J MAR SCI. VOL (4), APRIL 2014
bacterial communities in seagrass-bed sediments22,23
or associated with seagrass surfaces either roots or
shoots23-25. Whereas, studies centred on bacterial
communities inhabiting endophytically within the
seagrass tissues remain scarce with limited reports.
Prevalent endophytic bacterial communities
comprising Proteobacteria, Actinobacteria and
Firmicutes have been isolated from leaf tissues of
Thalassia testudinum26. Desulfovibrio zosterae has
been isolated from the surface- sterilized roots of
Z. marina27. Subsequently, Clostridium glycolicum
has been isolated from the rhizoplane and deep cortex
cells of Halodule wrightii 28 . More recently,
endophytic bacterial community of tissues of seagrass
Posidonia oceanica has been studied through cultureindependent approach using denaturing gradient gel
electrophoresis technique8, revealing presence of
bacterial endophytes that differed among locations
and tissue types. While it is obvious that certain
endophytic bacteria occur in seagrass tissues, the
scope and scale of those bacteria are largely unknown.
There are 13 species of seagrass found in Gulf of
Mannar and Palk Bay, among this Cymodocea
serrulata is the dominant species11. Previous studies
on Cymodocea serrulata have been concerned with
their extracts and bacteria associated with stem and
leaf for antibacterial activity against clinical
pathogens 29,30. Whereas, studies on diversity of
bacteria endophytically associated with Cymodocea
serrulata remains scarce. This study was undertaken
to isolate and define phylogenetic diversity of
bacterial population associated with roots of seagrass
Cymodocea serrulata. To obtain information about
the biological roles of root-associated bacteria, a
series of Plant Growth Promoting (PGP) traits were
assessed among the isolates.
Materials and Methods
Whole plant samples of the seagrass Cymodocea
serrulata were collected in February 2012 from
seagrass beds in the vicinity of Kasuwari Island, Gulf
of Mannar (fig. 1, Latitude 8° 52’ 18.0” N and
Longitude 78° 13’ 20.4” E). The collected samples
were placed in sterile plastic bag with a minimal
volume of seawater from the collection site and
Fig. 1 Map of the location of the sampling site, Kasuwari
Island, Gulf of Mannar.
transported with ice to the laboratory. Fresh healthy
plants with no sign of obvious injury were washed
with sterile seawater and processed within 24 h for
isolation of associated bacteria.
Seagrass sample was washed with sterile seawater
and roots were separated. Roots of the seagrass were
carefully cut with sterile knife and surface sterilized
with disinfectant solution containing 2% sodium
hypochlorite and 0.1% Tween 20. Samples were
rinsed with sterile milliQ water to remove disinfectant
and ground in sterile mortar and pestle. Ground
suspension was serially diluted and 0.1 mL of each
dilution was spread over Zobell Marine Agar medium
(Himedia, India) and natural sea water based isolation
medium IM131 contained 10 g of starch, 4 g of yeast
extract, 2 g of peptone and 18 g of agar in 1 L of
sterile seawater. Plates were incubated at 29°C for 1
to 6 weeks. Morphologically distinct colonies
obtained were further streaked onto same medium to
obtain pure cultures. Stock cultures were made in
Zobell Marine Broth with 20% sterile glycerol and
stored at - 20°C.
Effectiveness of the surface sterilization was
validated according to a previously described
method 32. Surface sterilized roots were soaked in
5 mL sterile milli Q and stirred for 1 min. The milli Q
(500 µL) was then spread onto Zobell Marine Agar
and incubated for 7 d at 29°C. Absence of bacterial
growth on the medium was an indication of good
surface-sterilization and successful elimination of
epiphytic bacteria.
The bacterial isolates were grown in Zobell
Marine Broth (Himedia, India) at 37°C for 12 to 72 h
and genomic DNA was extracted following standard
JOSE et al: MOLECULAR PHYLOGENY AND PLANT GROWTH
phenol-chloroform extraction procedure33. To amplify
bacterial 16S rDNA, a pair of eubacterial universal
primer was used: 27 F 5’-AGA GTT TGA TCC TGG
CTC AG-3’ and 1492R 5’- GGT TAC CTT GTT
ACG ACT T-3’34. PCR conditions for amplifying
bacterial 16S rDNA were as described earlier 35.
Amplification reaction was performed in thermal
cycler (MyCycler, Bio-Rad, USA) and the PCR
products were examined by electrophoresis through
1% agarose, stained with ethidium bromide, and
visualized on a UV transilluminator.
16S rDNA sequences of bacterial isolates have
been deposited in the Gen Bank database under the
accession numbers from KC012643 to KC012651.
The partial 16S rDNA sequences of the isolated
bacterial strains were initially analyzed and edited
by using BioEdit software 36 and the resulted
sequences were compared with those available in the
GenBank with BlastN algorithm. Sequences were
aligned using the multiple sequence alignment
program CLUSTAL X37 with default parameters and
the data were converted to PHYLIP format using
Bioedit. Phylogenetic tree was inferred by using
suitable programs of the PHYLIP (phylogeny
inference package) version 3.68 38 . Nucleotide
distances were calculated according to the algorithm
of Jukes and Cantor by using DNADIST and a
neighbour-joining tree was constructed using
NEIGHBOUR from the PHYLIP program package.
Bootstrap analysis was performed on 1000 random
samples taken from the multiple alignments.
In vitro screening of isolates for PGP traits
All the bacterial isolates were qualitatively
screened for various growth-promoting traits
according to Yadav et al.39 with minor modifications.
Bacterial strains were evaluated for their ability to
salubilize inorganic phosphate on Pikovskaya’s agar
plates40. In vitro indole acetic acid (IAA) production
was assayed based on the method described by
Hamdali et al. 41 . Fresh culture of isolates was
inoculated into 5ml of nutrient broth supplemented
with 3 mg/mL of L-tryptophan and incubated at 28°C
for 5 days. After incubation, the bacterial cells were
573
removed by centrifugation at 10,000 rpm for 10 min.
A drop of orthophosphoric acid and 2mL of
Salkowaski’s reagent was added to 1mL of the
supernatant and incubated at room temperature for
20 min. The development of pink colour was taken
as indication for the production of IAA. Production
of ammonia was screened in tubes containing 10 mL
of peptone water inoculated and incubated with fresh
cultures of isolates for 48 h at 30°C. After incubation,
about 0.5 mL of Nessler’s reagent was added to each
tube and observed for development of brown to yellow
colour, positive indication for ammonia production42.
Production of acetoin was tested among the isolates
using standard Voges-Proskauer test in 2 mL of MRVP broth 42 . The ability of bacteria to fix the
atmospheric nitrogen and to grow in nitrogen free
medium was tested on Jennson’s medium. Isolates
were spot inoculated and incubated at 28°C for 3 days.
Presence of growth on the medium was a positive
indication of nitrogen fixing activity43.
All the isolates were subsequently screened for
three extracellular enzymatic activities which possibly
support plant growth by degrading organic materials
associated with the sediments. Isolates were screened
qualitatively for amylase activity on starch agar
medium containing starch as carbon source. The
isolates were spot inoculated and incubated for 3 d at
28°C. After the incubation colonies were flooded with
iodine solution and left for 10 mins. Formation of
clear zone around the colonies was considered as
positive result for amylase activity44. Production of
protease was screened by spot inoculating the isolates
on nutrient agar supplemented with 10% skimmed
milk (w/v) and incubating for 2-4 d at 37°C. After
the incubation, the formation of the zone of clearance
around the colony was taken as an indication of
protease activity45. Isolates were screened for cellulase
activity by spot inoculating them on cellulase activity
indicator agar medium prepared with 0.5%
carboxylmethylcellulose (w/v) and incubating for
48-72 h at 37°C. After incubation, the plates were
flooded with 0.5% Congo red solution for 30 min,
rinsed with water, and then washed twice with 1 M
NaCl. Development of yellow halo around the
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INDIAN J MAR SCI. VOL (4), APRIL 2014
colonies against red background was taken as positive
indication of cellulose activity1.
Results
A total of 9 morphologically unique bacterial
isolates were obtained from roots of seagrass
Cymodocea serrulata. Among the two isolation
media employed for culturing seagrass associated
endophytic bacterial population, natural sea water
based isolation agar favoured isolation of 78% of
isolates while the Zobell marine agar favoured only
22%.
PCR amplification of 16S rDNA using a set of
universal eubacterial specific primers: 27 F and 1492R
yielded a single amplicon of ~1500 bp for all the
isolates. 16S rDNA of all the isolates were sequenced
and acquired sequences were in size range between
1379 bp and 1441 bp. In 16S rDNA based
phylogenetic tree (fig. 2), the isolates located in three
major clusters affiliated to Gammaproteobacteria,
Firmicutes and Actinobacteria.
The most abundant group of isolates was
affiliated with the phylum Proteobacteria
(Gammaproteobacteria), comprising two different
genera Vibrio and Photobacterium accounting for
45% of all bacterial isolates. Isolates JSA02, JSA05
and JAJ06 were found to be Vibrio species while
JAJ04 was identified as Photobacterium species.
Isolate JAS02 had 99.4% sequence similarity with
type strain Vibrio sagamiensis (GenBank AB
428909), luminous marine bacteria isolated from sea
water46. Other strains JSA05 and JSA06 fell in the
same clad in the phylogenetic tree with 99.9% and
99.8% sequence similarity respectively with Vibrio
owensii (GenBank GU018180), isolated from cultured
crustaceans in Australia47. Photobacterium species
JAJ04 shared maximum sequence similarity (99.9%)
with type strain Photobacterium rosenbergii
(GenBank AJ842344), associated with coral
bleaching48.
The second most dominant bacterial group in the
seagrass root associated endophytic bacterial
community was the phylum Firmicutes, comprising
two different genera Bacillus and Aerococcus
accounting for 33% of total population. Among the
9 bacterial isolates, 2 species belongs to the genus
Bacillus. Of these, strain JSA08 was clustered with
nearest type strain Bacillus gibsonii (GenBank
X76446) showing 99.5% similarity. Isolate JSA03
shared 99.8% sequence similarity with plant growth
promoting bacterium Bacillus methylotrophicus
(GenBank: EU194897), isolated from rice
rhizosphere49. Aerococcus strain JSA07 was 99.6%
identical to the type strain Aerococcus urinaeequi
(GenBank D87677).
Fig. 2 Neighbour-joining tree based on 16S rDNA sequences
of isolates, showing the phylogenetic relationship of
endophytic isolates and related genera. Bootstrap values are
expressed as percentages of 1000 replications. Bootstrap
values, >50 % are shown at branch points. Acidobacterium
capsulatumT (GenBank accession number CP001472)
The actinobacterial isolates belonged to the
genera Saccharomonospora and Kocuria representing
22% of entire isolates. Isolate JSA09 was identified
as Saccharomonospora species, shares 98.5%
sequence similarity with the type strain
JOSE et al: MOLECULAR PHYLOGENY AND PLANT GROWTH
575
Saccharomonospora glauca (GenBank Z38003)
isolated from moldy hay50. Another acinomycete
strain JSA01 was clustered with type strain Kocuria
rhizophila (GenBank Y16264) isolated from
rhizoplane of the narrow-leaved cattail51.
Saccharomonospora sp. JSA09 showed maximum
proteolytic activity whereas the bacilli strain, Bacillus
sp. JSA08 showed minimum activity. Production of
extracellular cellulase and amylase were detected in
8 (89%) and 6 (66%) isolates respectively.
In vitro screening of isolates for PGP traits
Discussion
All the 9 isolates were screened for their multiple
PGP traits and summarized in Table 1. Phosphate
solubilising ability was observed in 3 (39%) isolates.
Actinomycete isolate Kocuria sp. JSA01 showed
highest phosphate solubilisation activity with highest
zone diameter. Other isolates Saccharomonospora sp.
JSA09 and Vibrio sp. JSA05 showed moderate and
mild phosphate solubilising activities respectively.
Production of growth promoting substance indole
acetic acid was observed in 4 (44%) isolates, which
includes Kocuria sp. JSA01, Vibrio species JSA02,
JSA05 and JSA06. Ammonia production was detected
in 6 (62%) isolates. All the bacterial isolates were
found to be able to fix nitrogen and grew well in
nitrogen deficient medium. Acetoin production was
observed only with Vibrio sp. JSA06 while others
failed to produce it.
The present study provides the first report of
culture dependent endophytic bacterial communities
associated with roots of a seagrass, Cymodocea
serrulata from the Gulf of Mannar. In this study, 9
different endophytic isolates belonging to six different
bacterial genera have been isolated. Sequencing and
BLAST analysis of 16S rDNA of isolates revealed
the presence of high G+C Gram-positive, low G+C
Gram-positive and Proteobacteria groups among the
endophytic bacterial population. Presence of
specialized bacterial phylotypes in roots of seagrass
has previously been reported only through cultureindependent approach8. Phylogenetic analysis based
on 16S rDNA sequences assigned the isolates into
five genera; Vibrio, Photobacterium, Bacillus,
Aerococcus, Saccharomonospora and Kocuria,
categorised in three phyla: Gammaproteobacteria,
Firmicutes and Actinobacteria. Prevalence of
endophytic bacteria belong to Gammaproteobacteria,
Actinobacteria and Firmicutes has been observed in
Proteolytic activity was detected in 3 (38%)
endophytic bacterial isolates associated with
the seagrass roots. Actinomycete strain,
Table 1–Multiple plant growth promoting traits of endophytic bacterial community cultured from surface sterilized
roots of Cymodocea serrulata.
Strain
designation
Phosphate
IAA
Solublization Production
Kocuria sp.JSA01
Vibrio sp. JSA02
Bacillus sp. JSA03
Photobacterium
sp.JSA04
Vibrio sp.JSA05
Vibrio sp.JSA06
Aerococcus sp.JSA07
Bacillus sp. JSA08
Saccharomonospora
sp.JSA09
NH3
Production
Nitrogen
Fixation
Acetoin
production
Protease
activity
Cellulase
activity
Amylase
activity
+
-
+
+
-
+
+
+
+
+
+
+
-
+
-
+
+
+
+
+
-
+
+
+
+
ND
+
+
+
-
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+ Positive for PGP trait; - Negative for PGP trait; *ND – Not detected
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INDIAN J MAR SCI. VOL (4), APRIL 2014
leaf tissue of Thalassia testudinum26. Most dominant
group of endophytes found in the roots of Cymodocea
serrulata affiliated with Gammaproteobacteria
comprised two genera Vibrio and Photobacterium.
This dominance of Gammaproteobacteria is
consistent with previous reports on bacterial
communities associated with root surfaces of several
seagrass 24,25 . Furthermore, a recent report on
endophytic bacterial population associated with
seagrasss Posidonia oceanica also suggested
abundance of gammaproteobacteria in root tissues8.
Most of the endophytes isolated from root tissues of
Cymodocea serrulata shared high level 16S rDNA
sequence similarity with bacteria of rhizosphere or
marine ecosystem.
The endophytes tested in this study exhibited
multiple PGP traits that may promote plant growth.
Multiple PGP attributes of endophytic bacterial
population associated with seagrass roots are
consistent with views that bacterial-seagrass root
interactions are important to plant productivity52-54.
Phosphate solubilising bacteria ensure availability of
soluble phosphate to the plants by solubilising the
insoluble phosphate which is normally unavailable
for plants39. In this study, Saccharomonospora sp,
Kocuria sp and a Vibrio sp. were found to be positive
for phosphate solubilisation. Very recently, phosphate
solubilising bacteria have also been isolated from
seagrass rhizosphere soil. IAA is an important plant
growth hormone render positive effect on root growth
and morphology. In this study, four endophytes
showed positive result for IAA production and it is
consistent with earlier reports of IAA production in
seagrass-associated bacteria from other seagrass
Vallisneria americana 55. Moreover, established
seagrass tissue cultures have also been shown to
benefit from auxins56, 57. Capability to fix nitrogen is
widespread among many different bacteria isolated
from seagrass rhizosphere55. All the isolates tested in
this study exhibited nitrogen fixing ability. Presence
of nitrogen fixing bacteria has also been identified
among the endophytic population associated with
Posidonia oceanica through culture-independent
study8. Ammonia, acetoin, protease, amylase and
cellulase production also constitute a very important
part of plant growth promotion. The observed PGP
attributes are similar to that of bacteria associated with
terrestrial rhizosphere57, 59 and suggest that interactions
between bacteria and seagrass roots might be
similar to those described in better studied terrestrial
plants59-61, 39.
Conclusion
This is the first report of isolation and
identification of endophytic bacterial communities
associated with surface sterilized root tissues of
seagrass Cymodocea serrulata, suggesting the
presence of three phylotypes comprising diverse
bacterial genera in seagrass roots. PGP attributes of
the endophytic community reveals their possible
biological role within the root tissues of the seagrass.
Acknowledgements
Authors are grateful for the funding from
‘Research Fellowship in Science for Meritorious
Students Scheme’ of University Grant Commission,
India and also thankful to the UGC-MRP Scheme for
accessing the funded resource facilities in our
laboratory.
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