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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
DOI: http://dx.doi.org/10.1111/j.1574-6968.2012.02662.x 122-130 First published online: 1 November 2012

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.

Keywords
  • antimicrobial activity
  • Antipathes dichotoma
  • black coral
  • culturable microorganisms
  • diversity

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 (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; Toledo-Hernandez et al., 2007), which suggested that these microbes were part of the normal microbial communities. Furthermore, some coral diseases were believed to be 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 (Frias-Lopez 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 (Shnit-Orland & 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 double-layer technique with two marine pathogenic bacteria and two coral pathogenic fungi.

Materials and methods

Sample site and sample collection

Samples of three visually healthy colonies of the black coral A.dichotoma were collected at 5–10m 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 5–10cm 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.

The black coral Antipathes dichotoma and the positions of sample sites.

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. A 10-fold dilution was made and 0.1mL 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 1–4weeks 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 (gL−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 20g agar and 1L of seawater, and were adjusted to pH 7.0. For bacterial isolation, 0.05gL−1 streptomycin and potassium dichromate (50mL of 1gL−1 sterilized potassium dichromate in 1L 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, 0.5gL−1 benzylpenicillin and 0.03gL−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 1mL M2-broth medium (removed 20g agar from M2) and cultured at 30°C with shaking at 150r.p.m. for 3–5days. 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 12.5µL Taq premix (TakaRa, China), 1µL (10µM) of each primer (TakaRa), 1.5µL DMSO, 8µL water and 1µL of template DNA. After denaturation at 94°C for 6min, amplification was performed with 30 cycles of 40s at 94°C, 40s at 53°C, 2min at 72°C and a final extension at 72°C for 10min (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 (Toledo-Hernandez 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 5–14days 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 JQ647873JQ647893 and JQ647894JQ647903.

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 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, 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.

Neighbor-joining phylogenetic tree from analysis of >700bp 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.

Neighbor-joining phylogenetic tree from analysis of >500bp 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 neighbor-joining analysis of 1000 resampled datasets. Only values of >50% are shown. Scale bar: 0.05 substitutions per nucleotide position.

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 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.

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.

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 media-specific.

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 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 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.

The antimicrobial activity (double-layer assay) of several microbial isolates against marine bacterium Micrococcus luteus.

View this table:
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 isolatesMicrobial speciesMLPPASAV
SCSAAB0001Bacillus altitudinis10.28 ± 0.42
SCSAAB0007    B. amyloliquefaciens8.17 ± 0.3211.23 ± 0.56
SCSAAB0009    B. aquimaris8.06 ± 0.7111.28 ± 0.72
SCSAAB0010    B. aryabhattai
SCSAAB0011    B. flexus7.07 ± 0.1413.09 ± 0.46
SCSAAB0013    B. niabensis
SCSAAB0014    B. subtilis16.95 ± 0.6315.23 ± 0.59
SCSAAB0015    B. vallismortis
SCSAAB0017Thalassobacillus devorans
SCSAAB0018Micromonospora aurantiaca
SCSAAB0019    M. chokoriensis
SCSAAB0020    M. coxensis8.03 ± 0.8211.22 ± 0.848.12 ± 0.14
SCSAAB0021Novosphingobium panipatense
SCSAAB0022Paenibacillus glycanilyticus
SCSAAB0023Saccharomonospora xinjiangensis
SCSAAB0024Staphylococcus equorum11.41 ± 0.73
SCSAAB0028Streptomyces albogriseolus15.29 ± 0.4213.08 ± 0.657.56 ± 0.3911.87 ± 0.72
SCSAAB0029    S. albus11.29 ± 0.35
SCSAAB0030    S. labedae12.69 ± 0.44
SCSAAB0032    S. violascens
SCSAAB0035    S. xiamenensis16.25 ± 0.3816.94 ± 0.5614.33 ± 0.629.13 ± 0.30
SCSAAF0001Aspergillus ochraceopetaliformis17.15 ± 0.6911.62 ± 0.38
SCSAAF0006    A. sydowii
SCSAAF0009Fusarium proliferatum15.45 ± 0.49
SCSAAF0011Paecilomyces variotii
SCSAAF0013Penicillium chrysogenum9.28 ± 0.2811.41 ± 0.77
SCSAAF0015    P. citrinum8.18 ± 0.6114.00 ± 0.70
SCSAAF0017    P. oxalicum8.23 ± 0.21
SCSAAF0022Phoma putaminum
SCSAAF0023Microsphaeropsis arundinis
SCSAAF0024Myrothecium inundatum
  • 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.

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 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 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.

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 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, 2012AA092104), the Knowledge Innovation Program of Chinese Academy of Science (grant KSCX2-EW-G-12B), National Key Technologies R&D Program (grant 2011BAE06B04-03) 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.

References

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