Download Phylogenetic survey and antimicrobial activity of culturable

RESEARCH LETTER
Phylogenetic survey and antimicrobial activity of culturable
microorganisms associated with the South China Sea black coral
Antipathes dichotoma
Xiaoyong Zhang, Yulin Sun, Jie Bao, Fei He, Xinya Xu & Shuhua Qi
Key Laboratory of Marine Bio-resources Sustainable Utilization/Guangdong Key Laboratory of Marine Material Medical/RNAM Center for Marine
Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China
Correspondence: Shuhua Qi, Key
Laboratory of Marine Bio-resources
Sustainable Utilization/Guangdong Key
Laboratory of Marine Material Medical/RNAM
Center for Marine Microbiology, South China
Sea Institute of Oceanology, Chinese
Academy of Sciences, 164 West Xingang
Road, Guangzhou 510301, China. Tel.:
+86 20 8902 2112; fax: +86 20 8445 8964;
e-mail: [email protected]
Received 4 July 2012; revised 18 July 2012;
accepted 15 August 2012. Final version
published online 3 October 2012.
MICROBIOLOGY LETTERS
DOI: 10.1111/j.1574-6968.2012.02662.x
Editor: Paolina Garbeva
Keywords
antimicrobial activity; Antipathes dichotoma;
black coral; culturable microorganisms;
diversity.
Abstract
Most of our limited knowledge of microbes in corals comes from stony and
soft corals; the microbial diversity of black corals is still poorly understood.
Microbial diversity of the South China Sea black coral Antipathes dichotoma
was investigated using a culture-dependent method followed by analysis of bacterial 16S rRNA gene and fungal internal transcribed spacer sequences. A total
of 36 bacterial and 24 fungal isolates were recovered and identified, belonging
to three bacterial phyla (Firmicutes, Actinobacteria and Alphaproteobacteria) and
four fungal orders (Eurotiales, Hypocreales, Pleosporales and Botryosphaeriales).
The high level microbial diversity of A. dichotoma is in accordance with previous studies on those of some stony and soft corals. However, the lack of bacterial Gammaproteobacteria phylum in A. dichotoma is in sharp contrast to the
stony and soft corals, in which the Gammaproteobacteria phylum is relatively
common and abundant. Antimicrobial activities of 21 bacterial and 10 fungal
representative isolates (belonging to 21 different bacterial and 10 different fungal species, respectively) were tested against two marine pathogenic bacteria
and two marine coral pathogenic fungi. A relatively high proportion (51.6%)
of microbial isolates displayed distinct antibacterial and antifungal activities,
suggesting that the black coral-associated microorganisms may aid their host in
protection against marine pathogens. This is the first report on the diversity of
culturable microorganisms associated with black coral. It contributes to our
knowledge of black coral-associated microorganisms and further increases the
pool of microorganisms available for natural bioactive product screening.
Introduction
Coral reefs around the world are in decline, and infectious diseases are one of the main visible causes (Richardson & Aronson, 2002). As a result, more attention has
been focused on the coral-associated microbes that may
play a role in establishing diseases and the connections
existing between the microbial communities and the
overall health of the corals (Kellogg, 2004). In the last
four decades, great efforts have been made to identify
coral disease pathogens. These efforts routinely consisted
of microscopic observations of diseased coral tissues, all
of which revealed the presence of various bacteria and
fungi. The photosynthetic and heterotrophic bacteria
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(such as Phormidium corallyticum) were proposed as
potential agents of coral black band disease (Frias-Lopez
et al., 2004); and the bacterium Vibrio charcharii was
associated with coral white band disease (Richardson
et al., 1998). In addition, a few studies found that fungi
Aspergillus sydowii and Aspergillus versicolor were causal
agents of the coral aspergillosis (Nagelkerken et al., 1997;
Geiser et al., 1998; Fabricius & Alderslade, 2001; Sakayaroj et al., 2006). However, some microbes that had been
identified as potential agents of coral diseases have been
found in healthy corals (Koh et al., 2000; ToledoHernandez et al., 2007), which suggested that these
microbes were part of the normal microbial communities. Furthermore, some coral diseases were believed to be
FEMS Microbiol Lett 336 (2012) 122–130
123
Culturable microorganisms from Antipathes dichotoma
caused by microbial communities instead of a single pathogenic microbe (Zuluaga-Montero et al., 2010). These findings highlight our ignorance of the basic microbial ecology
of corals.
Most of our limited knowledge of microbes in corals
comes from stony and soft corals. From recent studies of
coral microbial ecology, it is known that microbes in
stony corals are distinct from those in the water column,
and there appear to be coral species-specific microbial
communities (Rohwer et al., 2001, 2002; Johnston &
Rohwer, 2007). Stony coral-associated microbes clearly
represent one of the most complex and important components of the biodiversity of coral communities (FriasLopez et al., 2002; Yakimov et al., 2006). Moreover, many
studies indicated that microbial communities occupy a
range of niches in stony corals, from within the surface
mucus layer (Bourne & Munn, 2005; Ritchie, 2006) to on
and within the coral tissue layers (Banin et al., 2000;
Frias-Lopez et al., 2002). In addition, microorganisms in
soft corals might be saprophytic or pathogenic, or may
provide other important functions for corals (Santavy &
Peters, 1997; Harvell et al., 1999). Microorganisms found
in soft corals may help the host by protecting them
against pathogens and/or may supply nutrients (ShnitOrland & Kushmaro, 2009).
Although our understanding of the microbial communities and their role in stony and soft corals is evolving,
the microbial diversity of black corals (order: Antipatharia)
is still poorly understood. This is mainly due to the
paucity of field studies that have focused on these black
corals, which can be found in all oceans at depths ranging
from those of shallow waters to 2000 or more meters
(Lapian, 2009). Ongoing investigation of the diversity and
antimicrobial activities of culturable microorganisms associated with the South China Sea corals, the black coral
Antipathes dichotoma, attracted our attention because the
coral harbors diverse and large microbial communities
and it is usually mistakenly viewed as a gorgonian coral
species.
This work aims to investigate the phylogenetic diversity
and antimicrobial activities of culturable microbial communities in the South China Sea black coral A. dichotoma,
which is unevenly distributed in the shallow waters of the
South China Sea (Zhou & Zhou, 1984; Su et al., 2008).
Eight different isolation media were utilized for microbial
isolation, and the phylogenetic diversities of the culturable bacteria and fungi associated with the black coral
were analyzed based on bacterial 16S rRNA gene and fungal internal transcribed spacer (ITS) sequences, respectively. In addition, the antimicrobial activities of the
microbial isolates were primarily assayed using a doublelayer technique with two marine pathogenic bacteria and
two coral pathogenic fungi.
FEMS Microbiol Lett 336 (2012) 122–130
Materials and methods
Sample site and sample collection
Samples of three visually healthy colonies of the black
coral A. dichotoma were collected at 5–10 m depth from
Sanya coral reef conservation (18°11′N, 109°25′E) in the
South China Sea, in August 2010. Replicate samples consisted of the outer 5–10 cm of a branch tip from separate
colonies dispersed over about a 1-km2 area of the coral
reef conservation, in order to account for small-scale spatial differences in the black coral microbial communities
and avoid sampling of coral clone mates (Kvennefors
et al., 2012). The three samples were transferred directly
to sterile plastic bags without seawater and then sent to
the laboratory as soon as possible, maintaining ice-cold
conditions to enable microbial isolation. The black coral
A. dichotoma sample and the positions of the sample sites
on the black coral are shown in Fig. 1.
Microbial isolation
The black coral samples were rinsed three times in sterile
seawater to remove transient and loosely attached microorganisms. The washed samples were then cut into 1-cm3
pieces and thoroughly homogenized using a sterile mortar
with the addition of two volumes of sterile seawater.
Fig. 1. The black coral Antipathes dichotoma and the positions of
sample sites.
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124
A 10-fold dilution was made and 0.1 mL of the resulting
solution was plated on different media plates (Zhang
et al., 2012). The inoculated plates were cultured at 26 °C
(for fungi) and 30 °C (for bacteria) for 1–4 weeks until
the morphology of the microorganisms could be determined. Microbial isolates were chosen and transferred
onto new separate agar plates on the basis of their morphological differences, based on visible examination of
growth characteristics. The resulting plates were incubated
at 26 °C (for fungi) and 30 °C (for bacteria) for pure
culture.
Four bacterial isolation media and four fungal isolation
media were used to isolate coral-associated bacteria and
fungi under aerobic conditions, respectively. The compositions of the eight media were as follows (g L 1): for
M1: glucose 4, yeast extract 4, malt extract 5; for M2:
mannitol 2, L-asparagine 0.1, CaCO3 2, K2HPO4 0.5,
MgSO4 0.1, FeSO4 0.001, vitamin B1 0.001, vitamin B6
0.001, vitamin lactoflavin 0.001, nicotinic acid 0.001, biotin 0.001, phenylalanine 0.001, alanine 0.0003; for M3:
peptone 5, yeast extract 3, NaCl 5; for M4: tryptone soy
15; for M5: glucose 10, peptone 1, starch 10, K2HPO4 1,
MgSO4 1; for M6: glucose 5, yeast extract 1, peptone 5;
M7: potato 200, glucose 20; for M8: starch 10, yeast
extract 5. All media contained 20 g agar and 1 L of seawater, and were adjusted to pH 7.0. For bacterial isolation, 0.05 g L 1 streptomycin and potassium dichromate
(50 mL of 1 g L 1 sterilized potassium dichromate in 1 L
sterilized media) was added to the bacterial isolation basic
media to inhibit the growth of fungi. For fungal isolation,
to inhibit the growth of bacteria, 0.5 g L 1 benzylpenicillin and 0.03 g L 1 Rose bengal were added to the fungal
isolation basic media.
DNA preparation and identification of
microbial isolates
For bacterial DNA extraction, the selected bacterial isolates
were inoculated into 7-mL centrifuge tubes containing
1 mL M2-broth medium (removed 20 g agar from M2)
and cultured at 30 °C with shaking at 150 r.p.m. for
3–5 days. Total genomic DNA was extracted from all
selected strains as described by Li & De (1995). From the
genomic DNA, nearly full-length 16S rRNA gene sequences
were amplified by polymerase chain reaction using primers
27°F (5′-GAGTTTGATCCTGGCTCAG-3′) and 1525R
(5′-AGAAAGGAGGTGATCCAGCC-3′; Warneke et al.,
2006). All of the primers were synthesized by SBS Genetech
(China). The polymerase chain reaction mixtures consisted
of 12.5 lL Taq premix (TakaRa, China), 1 lL (10 lM) of
each primer (TakaRa), 1.5 lL DMSO, 8 lL water and 1 lL
of template DNA. After denaturation at 94 °C for 6 min,
amplification was performed with 30 cycles of 40 s at
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X. Zhang et al.
94 °C, 40 s at 53 °C, 2 min at 72 °C and a final extension
at 72 °C for 10 min (Lee et al., 2003). Detailed information of fungal DNA extraction and fungal identification are
given by Zhang et al. (2012).
DNA sequencing of the selected bacterial and fungal
isolates was carried out by Invitrogen (China). Sequences
were corrected using SEQUENCHER, and the most similar
sequences in GenBank were found using Basic Local
Alignment Search Tool (BLAST) searches. When the top
three matching BLAST hits were from the same species and
were 98% similar to the query sequence, this species
name was assigned to the selected isolate (ToledoHernandez et al., 2008).
Determination of antimicrobial activity
The antimicrobial activities of bacterial and fungal isolates
were determined by a double-layer technique (Wu et al.,
2009). Selected bacterial and fungal isolates were grown
on M2 at 30 °C and M7 at 26 °C, respectively, for
5–14 days depending upon the growth rate of the various
isolates. Two marine bacteria (Micrococcus luteus and
Pseudoaltermonas piscida) and two marine coral pathogenic fungi [A. versicolor (AV) and A. sydowii (AS)] are
the indicator microorganisms for the double-layer assay.
Detailed information of the antimicrobial activity test is
given by Zhang et al. (2012).
Nucleotide sequence accession number
Bacterial 16S rRNA gene sequences of 21 representative
isolates and fungal ITS sequences of 10 representative isolates were deposited in GenBank/EMBL/DDBJ under
accession numbers of JQ647873–JQ647893 and JQ647894–
JQ647903.
Results
Isolation and phylogenetic analysis of
microbial communities
A total of 36 bacterial and 25 fungal isolates were recovered from the South China Sea black coral A. dichotoma
on the basis of their morphological differences. These
bacterial and fungal isolates were identify by bacterial 16S
rRNA gene sequences and fungal ITS sequences, respectively. By comparison with sequences in GenBank, the
sequences of all isolates shared 99–100% similarity with
their closest NCBI relatives, except that the fungal isolate
SCSAAF0025 (JQ647904) shared 93% similarity with the
known fungal species Gliomastix murorum YNS1116–4
(JQ354930) in GenBank. These identified isolates (including 36 bacterial and 24 fungal isolates) were assigned to
FEMS Microbiol Lett 336 (2012) 122–130
Culturable microorganisms from Antipathes dichotoma
three bacterial phyla: Firmicutes (35%), Actinobacteria
(23.3%) and Alphaproteobacteria (1.7%); and four fungal
orders: Eurotiales (30%), Hypocreales (6.6%), Pleosporales
(1.7%) and Botryosphaeriales (1.7%).
Further phylogenetic analysis was carried out on 21 bacterial and 10 fungal representatives (belonging to 21 different bacterial and 10 different fungal species, respectively),
which correspondingly showed similarity to 31 known
authentic species of bacteria and fungi. The results showed
that the 21 bacterial representatives belonged to 21 species
of eight genera (Fig. 2). Bacillus was the most diverse and
common genus, with eight species and 16 isolates in
the black coral A. dichotoma, followed by Streptomyces
(5 species and 10 isolates) and Micromonospora (3 species
and 3 isolates). The rest of the bacterial genera were rare,
125
occurring as singletons. The phylogenetic NJ tree of partial
ITS sequences of 10 fungal representatives is shown in
Fig. 3. Seven fungal genera were recognized from the 10
fungal isolates. The most abundant and diverse fungi were
observed in the genera Penicillium (3 species and 10
strains) and Aspergillus (2 species and 7 strains). Relatively
highly abundant (3 strains) fungi were detected in the
genus Fusarium. For the other four genera, only one isolate
was found.
Effect of isolation media on the recoverability
of microbial isolates
Four different media were selected for bacterial isolation
in this study. The results showed that the number and
Fig. 2. Neighbor-joining phylogenetic tree
from analysis of > 700 bp of 16S rRNA gene
sequences of bacteria isolated from the South
China Sea black coral Antipathes dichotoma.
The numbers at nodes are percentages
indicating the levels of bootstrap support,
based on a neighbor-joining analysis of 1000
resampled datasets. Only values of > 50% are
shown. Scale bar: 0.02 substitutions per
nucleotide position.
FEMS Microbiol Lett 336 (2012) 122–130
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126
X. Zhang et al.
Fig. 3. Neighbor-joining phylogenetic tree
from analysis of > 500 bp of ITS sequences of
fungi isolated from the South China Sea black
coral Antipathes dichotoma. The numbers at
nodes are percentages indicating the levels of
bootstrap support, based on a neighborjoining analysis of 1000 resampled datasets.
Only values of > 50% are shown. Scale bar:
0.05 substitutions per nucleotide position.
genera of recovered bacterial isolates differed for the four
media (Fig. 4). Bacteria could be recovered with all of the
four media; M2 yielded the highest number of bacterial
isolates and genera recovery with 14 isolates of seven genera. M3 had the least recoverability of bacterial isolates
(only six isolates). The Bacillus and Streptomyces isolates
were recovered from all four media. The genus Micromonospora could be only isolated from M2. The rest of the
bacterial genera were isolated in very small numbers.
Comparison of fungal isolates on four fungal isolation
media showed that the number and genera of recovered
fungal isolates also differed for the four types of media
(Fig. 4). M6, M7 and M8 had the most and same recoverability of fungal genera (four genera for each media),
whereas M5 yielded only two fungal genera. The genera
Aspergillus and Penicillium could be isolated from all four
media but Paecilomyces isolates were only isolated from
M6. The rest of the fungal genera were isolated in very
small numbers and cannot be concluded to be mediaspecific.
Distribution of microorganisms with
antimicrobial activity
All of the 21 bacterial and 10 fungal representatives
(belonging to 21 different bacterial species and 10 different fungal species, respectively) were tested against two
marine bacteria and two coral pathogenic fungi to
examine their spectrum of antimicrobial activity. Sixteen
isolates (51.6%) displayed antimicrobial activity against at
least one bacterium or fungus (Table 1). There were 11
and 5 antimicrobial isolates of bacteria and fungi,
respectively. Most antimicrobial isolates (12 of 16 isolates) exhibited distinct activity against marine bacterium
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Fig. 4. The number of bacterial and fungal isolates recovered on four
different bacterial and four fungal isolation media, respectively. Media
types marked by a letter (a) are bacterial isolation media; media types
marked by a letter (b) are fungal isolation media.
M. luteus. The antimicrobial activity (double-layer assay)
of several microbial isolates against M. luteus is shown in
Fig. 5. A few bacterial isolates (such as Streptomyces isolate SCSAAB0028 and SCSAAB0035) displayed relatively
high antimicrobial activity against all the four indicator
microorganisms. Bacillus subtilis isolate SCSAAB0014
exhibited strong activity against the two fungal indicators
A. versicolor and A. sydowii, and Streptomyces xiamenensis
isolate SCSAAB0035 displayed strong activity against the
two bacterial indicators. Among the 16 antimicrobial
FEMS Microbiol Lett 336 (2012) 122–130
127
Culturable microorganisms from Antipathes dichotoma
Table 1. Antimicrobial activity of culturable microbial representative isolates from the South China Sea black coral Antipathes dichotoma
Antimicrobial activity (zone of inhibition mm 1)
Microbial isolates
a
SCSAAB0001
SCSAAB0007a
SCSAAB0009a
SCSAAB0010a
SCSAAB0011a
SCSAAB0013a
SCSAAB0014a
SCSAAB0015a
SCSAAB0017a
SCSAAB0018a
SCSAAB0019a
SCSAAB0020a
SCSAAB0021a
SCSAAB0022a
SCSAAB0023a
SCSAAB0024a
SCSAAB0028a
SCSAAB0029a
SCSAAB0030a
SCSAAB0032a
SCSAAB0035a
SCSAAF0001b
SCSAAF0006b
SCSAAF0009b
SCSAAF0011b
SCSAAF0013b
SCSAAF0015b
SCSAAF0017b
SCSAAF0022b
SCSAAF0023b
SCSAAF0024b
Microbial species
ML
Bacillus altitudinis
B. amyloliquefaciens
B. aquimaris
B. aryabhattai
B. flexus
B. niabensis
B. subtilis
B. vallismortis
Thalassobacillus devorans
Micromonospora aurantiaca
M. chokoriensis
M. coxensis
Novosphingobium panipatense
Paenibacillus glycanilyticus
Saccharomonospora xinjiangensis
Staphylococcus equorum
Streptomyces albogriseolus
S. albus
S. labedae
S. violascens
S. xiamenensis
Aspergillus ochraceopetaliformis
A. sydowii
Fusarium proliferatum
Paecilomyces variotii
Penicillium chrysogenum
P. citrinum
P. oxalicum
Phoma putaminum
Microsphaeropsis arundinis
Myrothecium inundatum
10.28
8.17
8.06
—
7.07
—
—
—
—
—
—
8.03
—
—
—
—
15.29
11.29
12.69
—
16.25
—
—
—
—
9.28
8.18
8.23
—
—
—
PP
± 0.42
± 0.32
± 0.71
± 0.14
± 0.82
± 0.42
± 0.35
± 0.44
± 0.38
± 0.28
± 0.61
± 0.21
—
11.23
11.28
—
13.09
—
—
—
—
—
—
—
—
—
—
11.41
13.08
—
—
—
16.94
—
—
—
—
11.41
14.00
—
—
—
—
± 0.56
± 0.72
± 0.46
± 0.73
± 0.65
± 0.56
± 0.77
± 0.70
AS
AV
—
—
—
—
—
—
16.95
—
—
—
—
11.22
—
—
—
—
7.56
—
—
—
14.33
17.15
—
15.45
—
—
—
—
—
—
—
—
—
—
—
—
—
15.23
—
—
—
—
8.12
—
—
—
—
11.87
—
—
—
9.13
11.62
—
—
—
—
—
—
—
—
—
± 0.63
± 0.84
± 0.39
± 0.62
± 0.69
± 0.49
± 0.59
± 0.14
± 0.72
± 0.30
± 0.38
Each test was performed three times. Indicator bacteria: ML, Micrococcus luteus; PP, Pseudoaltermonas piscida. Indicator fungi: AV, Aspergillus
versicolor; AS, Aspergillus sydowii. Strong activity: zone of inhibition greater than 15 mm. Moderate activity: zone of inhibition between 10 and
15 mm. Weak activity: zone of inhibition < 10 mm. —, traces or no antagonistic effects were observed. Microbial isolates marked by a letter (a)
are bacterial isolates; microbial isolates marked by a letter (b) are fungal isolates.
active isolates, the bacterial genera Bacillus and Streptomyces, and fungal genus Penicillium isolates had the highest
proportions of antimicrobial activity: 16.1%, 12.9% and
9.7%, respectively.
Discussion
The present study provides the first analysis of the microbial communities inhabiting black coral species using culture-dependent techniques. All 21 bacterial and 10 fungal
species were isolated from the South China Sea black coral
A. dichotoma. The high level of microbial diversity in
A. dichotoma is in accordance with previous studies on those
of stony coral Acropora digitifera from the Gulf of Mannar
and some soft corals (Harder et al., 2003; Gray et al.,
2011). However, the lack of bacterial Gammaproteobacteria
phylum in A. dichotoma is in sharp contrast to the stony
FEMS Microbiol Lett 336 (2012) 122–130
and soft corals, in which the Gammaproteobacteria phylum
is relatively common and abundant (Harder et al., 2003;
Nithyanand & Pandian, 2009; Gray et al., 2011). This is
probably due to the different morphological structures of
the black coral A. dichotoma and stony and soft coral species, or possibly that Gammaproteobacteria phylum are not
trapped in the tissues of A. dichotoma.
The Firmicutes phylum was the largest bacterial group
in A. dichotoma, and most species (such as B. altitudinis,
B. amyloliquefaciens and B. vallismortis) of Firmicutes
phylum in A. dichotoma were not recovered from stony
and soft corals (Harder et al., 2003; Lampert et al., 2006;
Nithyanand & Pandian, 2009; Nithyanand et al., 2011).
Although a few studies have reported that independently
cultured Actinobacteria was commonly associated with
stony and soft coral species (Webster & Bourne, 2007;
Castro et al., 2010), little is known about the culturable
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128
Fig. 5. The antimicrobial activity (double-layer assay) of several
microbial isolates against marine bacterium Micrococcus luteus.
actinobacteria associated with corals (Lampert et al., 2006;
Nithyanand & Pandian, 2009; Gray et al., 2011; Nithyanand et al., 2011). In this study, the actinobacterial species
Saccharomonospora xinjiangensis and alphaproteobacterial
species Novosphingobium panipatense were first isolated
from corals.
Fungi in corals are now known to cause coral diseases,
but little attention has been paid to the nature of fungal
communities in corals. In this study, a relatively diverse
fungal community (24 isolates of 10 fungal species) was
found in A. dichotoma. Highly diverse fungal communities
also have been found in many different soft coral species
collected from Raffles Lighthouse in Singapore (Koh et al.,
2000) and the Caribbean (Toledo-Hernandez et al., 2007,
2008). However, the fungal community compositions were
obviously different in different coral species; most fungal
species isolated from A. dichotoma were not found in soft
corals from Raffles Lighthouse in Singapore (Koh et al.,
2000) and the Caribbean (Toledo-Hernandez et al., 2007,
2008). In the present study, all fungal isolates were
identified as known fungal species except for the strain
SCSAAF0025 (JQ354930), which might be a candidate for a
new species or genus. Aspergillus and Penicillium were the
most diverse and common genera (17 of 24 isolates). The
two genera have also been found frequently in stony corals
(Priess et al., 2000), soft corals (Zhang et al., 2012) and
other marine invertebrates such as sponges (Holler et al.,
2000; Zhou et al., 2011). It appears that the two fungal
genera are successful at colonizing different hosts and are
ubiquitous in many marine organisms.
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X. Zhang et al.
The results (Fig. 4) clearly indicate that different media
yield different numbers and species of microbial isolates
in the black coral A. dichotoma. For the four bacterial isolation media used in this study, M2 had the best recoverability of bacterial genera, and could recover all eight
bacterial genera except for Novosphingobium, which was
only isolated from M3. Compared with the other three
media, M2 contains lower concentrations of several free
amino acids and vitamins. Gil et al. (2009) reported that
the best nitrogen sources for bacterial isolation were proteins, peptones and amino acids. Our results support the
notion that diverse bacteria can be well recovered on
media with low concentrations of free amino acids, and
also indicate that vitamins may play important roles in
the isolation of bacteria from black corals. A combination
of M2 and M3 would be sufficient for isolating bacteria
from the black coral A. dichotoma in this study.
Of the four fungal isolation media tested in this study,
M6, M7 and M8 were equally suitable for culturing a
similar diversity of fungi with different numbers of isolates. Koh et al. (2000) reported that a combination of
only two media (GYA and PDA, corresponding to M6
and M7 in this study) would be sufficient for isolating
fungi from soft corals. However, a well designed isolation
protocol with multiple isolation media was essential for
isolating diverse and abundant fungi from the black coral
in this study.
On investigating the antimicrobial activity of culturable
microorganisms in the black coral A. dichotoma against
two marine pathogenic bacteria and two coral pathogenic
fungi, 51.6% of isolates displayed antimicrobial activity
against at least one bacterium or fungus (Table 1), suggesting that the culturable microorganisms could fend off
or develop resistance to certain microbial diseases of the
black corals. These results concur with a few previous
reports stating that 20–70% of culturable microorganisms
in stony and soft corals exhibited antimicrobial activity
(Nithyanand & Pandian, 2009; Shnit-Orland & Kushmaro,
2009).
Of the above 16 antimicrobial isolates, the bacterial
genus Bacillus had the highest proportion of antimicrobial
activity, and B. subtilis isolate SCSAAB0014 exhibited
strong activity against two fungal indicators, A. versicolor
and A. sydowii, which supported the hypothesis that
Bacillus sp. might play a protective role in the coral hosts
(Nithyanand & Pandian, 2009). The Bacillus genus is an
important antibiotic resource. Over 800 antibiotic metabolites, including the important group of peptide antibiotics such as bacitracin, gramicidin and polymyxin B, are
produced by various Bacillus sp. Two Streptomyces isolates, SCSAAB0028 and SCSAAB, displayed relatively
strong antimicrobial activities against all the four indicator microorganisms tested, suggesting that members of
FEMS Microbiol Lett 336 (2012) 122–130
Culturable microorganisms from Antipathes dichotoma
the genus Streptomyces in A. dichotoma had a broad antimicrobial spectrum. Three members of the genus Penicillium here exhibited distinct antibacterial activity against
the two bacterial indicators, ML and PP, which agreed
with the opinion that Penicillium genus produces antibacterial compounds (Tejesvi et al., 2011). For example,
Wang et al. (2012) found three new aromatic polyketides
isolated from the fermentation broth of the associated
gorgonian-associated fungus Penicillium commune which
showed moderate antimicrobial activities against Escherichia coli and Enterobacter aerogenes. In summary, many
culturable microbial species had potential antimicrobial
properties in this study, e.g. B. subtilis, S. albogriseolus,
S. xiamenensis, and P. chrysogenum have been reported to
produce antimicrobial compounds (Feio et al., 2004; Cui
et al., 2007; Onyegeme-Okerenta et al., 2009; Xu et al.,
2012), which further supports our proposal that black
coral-associated microorganisms need to be investigated
for bioactive compounds.
Acknowledgements
The authors are grateful to the National Basic Research
Program of China (grant 2010CB833803), the National
Natural Science Foundation of China (grant 40931160435
and 40976090), National High Technology Research and
Development Program of China (863 Program, 2012A
A092104), the Knowledge Innovation Program of Chinese
Academy of Science (grant KSCX2-EW-G-12B), National
Key Technologies R&D Program (grant 2011BAE06B0403) and Guangdong Natural Science Foundation of
China (grant S2011040000144) for financial support. The
authors are grateful to Dr Hui Huang and Xiubao Li
(South China Sea Institute of Oceanology, Chinese Academy of Sciences) for their kindness in identifying the
black coral samples.
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