Fungal Diversity DOI 10.1007/s13225-012-0192-7

Phylogenetically diverse endozoic fungi in the South China Sea sponges and their potential in synthesizing bioactive natural products suggested by PKS gene and cytotoxic activity analysis

Zhisheng Yu & Baohua Zhang & Wei Sun & Fengli Zhang & Zhiyong Li

Received: 20 June 2012 /Accepted: 20 July 2012 # Mushroom Research Foundation 2012

Abstract Sponges are well documented to harbor large as the predominant component in the culturable amounts of microbes. Though it is known that sponge- fungal community. Particularly, genera Schizophyllum, derived fungi are important sources for marine natural prod- Sporidiobolus, and Bjerkandera in phylum Basidiomycota ucts, the phylogenetic diversity and biological function of and Yarrowia in phylum were isolated sponge-associated fungi remain largely unknown. In this from marine sponges for the first time. PKS genes were study, the diversity of culturable endozoic fungi in sponges detected in 12 isolates suggesting their potential for synthe- from the South China Sea was revealed based on the ITS sizing PKS compounds. Among the 12 isolates with PKS phylogenetic analysis. Meanwhile the fungal potential for genes, 9 isolates displayed strong in vitro cytotoxic activity producing bioactive natural products was estimated accord- (e.g. IC50<50 μg/ml) against human cancer cell lines A- ing to the detection of Beta-ketosynthase in the polyketide 549, Bel-7402, A-375 and MRC-5. This study demonstrates synthase (PKS) gene cluster and cytotoxic activity bioassay. the phylogenetically diverse endozoic fungi in South China As a result, diverse fungi including 14 genera (Aspergillus, Sea sponges, and highlights the potential of sponge- Penicillium, Scolecobasidium, Eurotium, Alternaria, Fusa- associated fungi in producing biologically active natural rium, Hypocreales, Yarrowia, Candida, Hypoxylon, Spori- products. diobolus, Schizophyllum, Bjerkandera, and Trichosporon) in ten orders (Xylariales, Moniliales, Pleosporales, Saccha- Keywords Cytotoxic activity . Fungal diversity . Polyketide romycetales, Hypocreales, , Sporidiobolales, synthase (PKS) gene . Sponge Agaricales, Aphyllophorales and Tremellales) of phyla Ascomycota and Basidiomycota were isolated with Introduction Zhisheng Yu and Baohua Zhang contributed equally to this paper. Z. Yu : W. Sun : F. Zhang : Z. Li Since the pioneering work of Barghoorn and Linder (1944), Marine Biotechnology Laboratory, State Key Laboratory significant progress has been made on marine-derived fungi of Microbial Metabolism and School of Life Sciences (Bugni and Ireland 2004; Raghukumar 2008; Debbab et al. and Biotechnology, Shanghai Jiao Tong University, 2011; Jones 2011). However, compared with their terrestrial Shanghai 200240, People’s Republic China counterparts, marine fungi remain one of the most under B. Zhang explored groups in the marine environment, especially ma- Eastern Hepatobiliary Surgery Hospital, rine fungi associated with marine macro-organisms (Wang Second Military Medical University, et al. 2008; Li and Wang 2009). Shanghai 200438, People’s Republic China As the oldest animal e.g. over 600 million years old, Z. Li (*) marine sponges (Porifera) form close association with a School of Life Sciences and Biotechnology, wide variety of microorganisms including bacteria, archaea Shanghai Jiao Tong University, and fungi (Hentschel et al. 2006; Taylor et al. 2007a, b; Gao 800 Dongchuan Road, Shanghai 200240, People’s Republic China et al. 2008; Liu et al. 2010; Ding et al. 2011; Lee et al. 2011; e-mail: [email protected] Paz et al. 2010; Zhou et al. 2011; Schmitt et al. 2012; Fungal Diversity

Webster and Taylor 2012). In contrast to the understanding Cinachyrella australiensis (3–10), Diplastrella megastellat of the sponge bacterial diversity (Lee et al. 2011; Webster (1–9) and Geodia neptuni (1–11) were collected nearby and Taylor 2012; Schmitt et al. 2012), little is known about Yongxin Island and LingShui in the South China Sea at a the fungal diversity due to the lack of direct evidence of depth of ca.10–20 m. Sponges were transferred directly to fungal mycelia, for example, microscopic, immunological, zip-lock bags containing sea water to prevent the contact of or fluorescence in situ hybridization detection. At present, sponge tissue with the air. The samples were transported to studies on the fungal diversity in sponges are relatively few the laboratory on ice and processed immediately for fungal than those addressing the metabolic versatility of sponge- isolation. derived fungi (Lang et al. 2007; Proksch et al. 2008; Xie et Before the isolation of endozoic fungi, sponge sample al. 2008; Abdel-Lateffa et al. 2009; Zhang et al. 2009a; Lee was cut into small pieces and rinsed three times with sterile et al. 2010). Meanwhile, the biological and ecological func- artificial seawater (ASW) (Li and Liu 2006) to get rid of tions of sponge-associated fungi remain largely unanswered fungi on the sponge surface and in the sponge inner cavity. (Maldonado et al. 2005; Rot et al. 2006; Baker et al. 2009; Sponge inner issue was cut into pieces (1–3cm3) with a Paz et al. 2010). sterile scalpel and immersed in sterile calcium-and- Fungi associated with sponges display diverse biological magnesium-free ASW for 10 min. Then, the small sponge activities and represent the single most prolific source for pieces were homogenized by a high-speed pulse-type ho- marine fungi-derived bioactive compounds (Proksch et al. mogenizer machine. The homogenate was diluted with ster- 2003, 2008, 2010; Bugni and Ireland 2004; Amagata et al. ile ASW at three dilutions (1:10, 1:100, 1:1000). One 2006a; Bhadury et al. 2006; Lang et al. 2007; Saleem et al. hundred micro liters of each dilution was plated onto Martin 2007; Xie et al. 2008; Abdel-Lateffa et al. 2009; Lee et al. and MYPG media (Ding et al. 2011) in triplicate and incu- 2010; Wiese et al. 2011), suggesting their importance in the bated at 28 °C for 7–15 days. All the media were prepared development of marine drugs. Polyketides have been im- with ASW and adjusted to pH 7.4–7.6. Media were auto- mensely concerned over the past decades. Various novel claved at 121 °C for 20 min except the medium containing polyketide compounds with biological activities and ecolog- glucose which was autoclaved at 115 °C for 30 min. Thirty ical functions have been found from marine-derived micrograms per liter of streptomycin and ampicillin were microbes (Blunt et al. 2009). The presence of polyketide added to the media to inhibit bacterial growth. synthase (PKS) in sponge-associated fungi suggests the potential in producing related polyketide compounds and Genomic DNA extraction, PCR amplification of rDNA-ITS guides the production of related natural products. The filtra- fragment and phylogenetic analysis tion of PKS genes from sponge-associated bacteria has started (Schirmer et al. 2005; Kim and Fuerst 2006; Jiang After cultivation in Martin medium at 180 rpm, 28 °C for 5– et al. 2007; Hochmuth and Piel 2009; Zhang et al. 2009b, c; 7 days, the cultures were centrifuged at 5,000×g for 5 min. Siegl and Hentschel 2010), but until now, the screening of The precipitate was grinded in a mortar containing 600 μl PKS genes in sponge-associated fungi is still very scarce CTAB lysis buffer (2 % CTAB, 1.4 M NaCl, 100 mM Tris, except our recent report (Zhou et al. 2011). 20 mM EDTA, 1 % PVP). The mycelial mixture was trans- In this study, the culturable endozoic fungal associated ferred into a 1.5-ml Eppendorf tube and heated to 65 °C for with marine sponges in the South China Sea were isolated 30 min, extracted twice with an equal volume of phenol/ and their phylogenetical diversity was investigated using chloroform/isoamyl alcohol (25:24:1) and washed with ITS phylogenetic analysis. Meanwhile, their potential for chloroform/isoamyl alcohol (24:1). After centrifugation at producing bioactive natural products was evaluated accord- 10,000×g for 5 min, the supernatant was transferred to a ing to the detection of the conserved Beta-ketosynthase of new microtube and precipitated by adding equal volume of polyketide synthase (PKS) gene and the bioassay of cyto- isopropanol at −20 °C for 1 h. Finally, the DNA pellets were toxic activity. collected by centrifugation (12,000×g, 15 min), washed with 75 % ethanol twice and re-suspended in 40 μlTE Buffer (10 mM Tris, 1 mM EDTA, pH 8.0). RNA was Materials and methods removed by adding 2 μl of RNase A (10 mg/ml; Invitrogen) at 60 °C for 10 min. Sponge sampling and isolation of sponge-associated fungi The resulting genomic DNA solution was used as a template to amplify the fungal rDNA-ITS fragment (500– Ten known marine sponges Amphimedon queens- 800 bp) using the primers ITS1 and ITS4 (White et al. landica (2–1), Holoxea sp. (3–1), Phyllospongia foliascens 1990). The PCR reaction mixture (40 μl) was compose of (3–2), Iotrochota sp. (3–3), Ircinia felix (3–5), Aplysina Premix Taq 20 μl (TaKaRa Taq 1.25U/25 μl, dNTP Mixture aerophoba (3–6), Xestospongia testudinari (3–7), 2×conc.; 0.4 mM, PCR Buffer 2×conc.; 3 mM Mg2+), 1 μl Fungal Diversity of each primer (10 μM), 1 μl of fungal DNA, 2 μl DMSO proteins in the nr protein database using the BLASTP algo- and 15 μl of ddH2O. PCR was carried out as follows: initial rithm. Unrooted phylogenetic tree based on amino acid denaturation (94 °C for 5 min), 30 cycles of denaturation sequences of KS domain was constructed using neighbor- (94 °C for 30 s), primer annealing (56 °C for 30 s), and joining method in MEGA 4.0 combined with bootstrap elongation (72 °C for 1 min), with a final longation at 72 °C analysis with 1,000 replications. for 10 min. PCR products were purified using Cycle-pure Kit (Omega) according to the manufacturer’s instructions. Cytotoxic activity assay of sponge endozoic fungi The library of ITS sequences was constructed using the pEASY–Blunt Cloning kit based on blue-white screening Fungal isolates with PKS genes were grown in the 1 L flask method (TransGen) following the manufacturer’sinstructions. with 500 ml Martin medium on a rotary shaker at 180 rpm, White transformants were inoculated into LB broth (with 28 °C for 3–7 days. The fermentation broth was extracted 100 μg/ml ampicillin) and incubated overnight at 37 °C. The with ethyl acetate. The extracts were concentrated under positive clones with the target fragments were identified and vacuum at 50 °C. sequenced using vector M13F/R primers on ABI 3730xl For the in vitro cytotoxic activity assay, the obtained capillary sequencers (Applied Biosystems). dried extracts were dissolved in methanol with a final con- Fungal rDNA-ITS sequences were aligned on the SILVA centration of 10 mg/ml and tested at five concentration database (http://www.arb-silva.de/aligner/) to identify the gradients (100 μg/ml, 25 μg/ml, 6.25 μg/ml, 3.125 μg/ml, sequence similarity to reference (Pruesse et al. 2007). The and 1.5625 μg/ml). Cisplatin was used as positive control at most appropriate relative sequences were selected and five concentration gradients (20 μg/ml, 10 μg/ml, 5 μg/ml, imported into MEGA 4.0 (Tamura et al. 2007). The unrooted 1 μg/ml, and 0.1 μg/ml). The tetrazolium–based colorimet- phylogenetic tree was constructed using neighbor-joining ric method, i.e. MTT [3-(4,5-dimethythiazoyl-2-yl) 2,5- method combined with bootstrap analysis setting with 1,000 diphenylte-trozoliumbromide] colorimetric analysis, was replications. used for the bioassay of cytotoxicity against human lung carcinoma cell line (A-549), human liver carcinoma cell line PKS genes screening and phylogenetic analysis (Bel-7402), human melanoma carcinoma cell line (A-375) and human normal embryo lung fibroblasts cell line (MRC-5). Primer pairs LC1/LC2c (5′-GATCGTTGGATCCTCTA/3′- Well-growing carcinoma cells were collected and seeded TAAGATCTCGAGCTCTAGA) and LC3/LC5c (5′- in 96-well plates at 1×105/ml density. When the cells an- GATCGTTGGATCCTCTA/3′-TAAGATCTCGAGCTC- chored to the plates, the culture medium was replaced with TAGA) designed for the highly conserved sequences of β- fresh medium containing various concentrations of the fun- ketoacyl synthase (KS) domain was used to detect PKS gene gal extracts. Three duplicate wells were used for each sam- in the fungal isolates (Bingle et al. 1999). The PCR mixture ple. After incubation at 37 °C, 5 % CO2 for 48 h, 50 μl1× (50 μl) contained 1.5 μl template DNA, 25 μl Premix Taq MTT was added to each well for another 4 h incubation. 20 μl (TaKaRa Taq 1.25U/25 μl, dNTP Mixture 2×conc.; Then, the MTT medium was discarded and warm dimethyl- 0.4 mM, PCR Buffer 2×conc.; 3 mM Mg2+), 2 μl primer sulfoxide (DMSO) 150 μl was added. Absorbance was

LC1, 2 μl primer LC2c,and 19.5 μl ddH2O. The PCR measured at 520 nm. The inhibition rate (IR%) was calculated reaction was as follows: 4 min at 95 °C; 35 cycles of 30 s as follows: IR%0(ODcontrol−ODsample )/ODcontrol×100 %. at 95 °C, 1 min at 50 °C, 2 min at 72 °C, and 7 min at 72 °C. The PCR product was purified with Cycle-pure Kit (Omega) Nucleotide sequence accession number and transformed into Trans1-T1 Phage-Resistant Chemical- ly Competent Cell using pEASY-Blunt Simple Cloning Kit Fungal rDNA-ITS sequences obtained in this study were (Transgen). The positive recombinants were screened on deposited in GenBank under accession numbers JQ697508- plates with X-Gal, IPTG and ampicillin by color-based JQ697555; Fungal PKS gene sequences were submitted to recombinant selection method. Positive clones were se- GenBank under accession numbers JQ697496-JQ697507. quenced using vector primers M13F and M13R on ABI 3730xl capillary sequencer by Shanghai Invitrogen Compa- ny. The obtained PKS gene sequences were analyzed with Results BLASTX and aligned against reference sequences by CLUSTAL X (Thompson et al. 1997). Translated protein Phylogenetic diversity of sponge-associated cultivable sequences were derived from nucleotide sequences using endozoic fungi the ORF FINDER available at the NCBI website (http:// www.ncbi.nlm.nih.gov/projects/gorf/). The deduced amino In total, 48 fungal isolates were obtained, among which 11 acid sequences were used as queries to search the related isolates were from Geodia neptuni, 9 from Amphimedon Fungal Diversity queenslandica and 8 from Diplastrella megastellat,7from Sporidiobolales,andTremellales were isolated successfully Cinachyrella australiensis, 5 were from Ircinia felix, no fungal from the South China Sea sponges. All the isolates showed isolates were recovered from Aplysina aerophoba, the rest 99–100 % similarity to their closest relatives. As shown in 8 isolates were from the other four sponges. Based on the Fig. 1, most of the fungal isolates showed close affinity to the BLAST analysis using the rDNA-ITS sequences of the 20 other marine-derived relatives. representative fungi selected according to the morphological characteristics, the culturable fungal community was assigned Screening of PKS genes in sponge-associated cultivable to Ascomycota (87.5 %) and Basidiomycota (12.5 %), embrac- endozoic fungi ing 14 genera (Aspergillus, Penicillium, Scolecobasidium, Eurotium, Alternaria, Fusarium, Hypocreales, Yarrowia, Can- Among the 48 isolates, KS domain (ca. 686 bp) was dida, Hypoxylon, Sporidiobolus, Schizophyllum, Bjerkandera, detected in 12 isolates by LC3/LC5 primers (10) or LC1/L and Trichosporon) in ten orders (Pleosporales, Sporidiobo- C2c primers (2). The KS positive fungi belong to Aspergil- lales, Eurotiales, Hypocreales, Saccharomyceales, Xylariales, lus (9), Penicillium (2) and Eurotium (1) in order Eurotiales. Agaricales, Aphyllophorales, Tremellales and Moniliales) Based on the BLAST analysis (Table 2), the KS sequences (Table 1). Eurotiales was the dominant group (relative abun- of isolates MD7_8, MD11_3, MD11_4, MY11_4, MD32_7, dance 54.2 %) in the cultivable fungal community, among MD7_1, MD10_1 and MY11_8 show 100 % sequence which Aspergillus was predominant with 26/48 isolates. similarity to 6-methylsalicylic acid synthase of Aspergillus Though Gao et al. (2008) have reported the presence of a few Terreus. The KS sequences of MY5_ 3 and MY5_400 are uncultured Basidiomycota in the marine sponges Suberites 99 % similar to nonreducing polyketide synthase in Peni- zetekiand and Mycale armata using culture-independent cillium janthinellum. In contrast, KS sequences of isolates molecular strategy, Basidiomycota have rarely been isolated MD10_2 and MY21_10 show novelty because of their successfully from sponges compared with Ascomycota (Liu et relatively low similarity to their closest relatives, i.e. 91 % al. 2010; Menezesa et al. 2010; Paz et al. 2010;Dingetal. homology with polyketide synthase of Aspergillus ochra- 2011; Thirunavukkarasu et al. 2012). In this study, four ceus and 80 % homology with polyketide synthase of As- basidiomycete fungal taxa Agaricales, Aphyllophorales, pergillus clavatus, respectively. The phylogenetic analysis

Table 1 The phylogenetic affiliations of fungi associated with sponges

Representative Taxon Closest relative (accession no.) Identity Source Frequency (accession no.) (%) isolated Phylum Order

MD10_2 (JQ697527) Ascomycota Eurotiales Aspergillus insulicola (EF661430) 99 plant 1 MY9_1 (JQ697534) Aspergillus penicillioides (HQ702383) 99 marine 2 MY5_0 (JQ697509) Aspergillus terreus (FJ571438) 99 marine 22 MD21_17 (JQ697553) Aspergillus oryzae (GU385811) 99 marine 1 MY5_3 (JQ697529) Penicillium janthinellum (HQ839782) 99 soil 2 MD21_16 (JQ697524) Penicillium steckii (DQ123665) 99 plant 1 MY21_10 (JQ697548) Eurotium rubrum strain (HQ316568) 100 marine 1 MY5_4 (JQ697530) Moniliales Scolecobasidium sp. OUCMBIII101045 100 marine 1 (HQ914903) MY9_3 (JQ697510) Pleosporales Alternaria alternata (AB667801) 100 human 2 MD3_1 (JQ697514) Hypocreales Fusarium solani (AM412637) 99 soil 1 MD10_3 (JQ697517) Hypocreales sp. KH00296 (GU017511) 100 marine 2 MD21_15 (JQ697523) Yarrowia lipolytica (EF197819) 99 marine 1 MD32_4 (JQ697525) Candida parapsilosis (EF190230) 100 marine 2 MY9_2 (JQ697535) Candida sp. D3978 (AM491365) 99 insect 1 MY11_12 (JQ697543) Candida glaebosa (FM178351) 100 marine 1 MY11_2 (JQ697538) Xylariales Hypoxylon fragiforme (JN390828) 99 plant 1 MD21_1 (JQ697550) Basidiomycota Sporidiobolales Sporidiobolus pararoseus (HQ379160) 99 plant 1 MY11_1 (JQ697513) Agaricales Schizophyllum commune (AF280758) 100 marine 3 MD31_3 (JQ697552) Aphyllophorales Bjerkandera adusta (JN182863) 99 soil 1 MY21_9 (JQ697555) Tremellales Trichosporon montevideense (GU299461) 100 effluent 1 Fungal Diversity

Fig. 1 Neighbor-joining unrooted phylogenetic tree constructed using bar represents 0.2 nucleotide substitutions per site. The black cycle rDNA-ITS sequences (ca.700 bp) of cultured fungi from 10 sponges in represents nucleotide sequences obtained in this study. The white cycle the South China Sea. Number at branch indicates Bootstrap value represents the closest relative associated with the marine environment (>50 %) of neighbor-joining analysis from 1,000 replicates. The scale Fungal Diversity

Table 2 Similarity to the closest relatives in GenBank of KS PKS fragment Closest relative (accession no.) Identity (%) amino acid sequences of isolates from sponges MD7_8 (JQ697496) Aspergillus terreus 6-methylsalicylic acid synthase (BAA20102) 100 MD11_3 (JQ697497) 100 MD11_4 (JQ697498) 100 MY11_4 (JQ697499) 100 MD32_7 (JQ697501) 100 MD7_1 (JQ697504) 100 MD10_1 (JQ697505) 100 MY11_8 (JQ697500) 100 MY5_3 (JQ697502) Penicillium janthinellum nonreducing 99 MY5_400 (JQ697503) polyketide synthase (ADY75762) 99 MD10_2 (JQ697506) Aspergillus ochraceus MSAS-type polyketide synthase (AAS98200) 91 MY21_10 (JQ697507) Aspergillus clavatus NRRL 1 6-methylsalicylic acid synthase MsaS 80 (XP_001273093) showed that most of sponge fungal KS sequences gathered the 12 fungi displayed moderate to strong in vitro anti-tumor with type I PKS sequences (Fig. 2). activity against the 4 human cancer cell lines. Particularly, 8 Aspergillus terreus strains (MD11_3, MD11_4, MY11_4, Cytotoxic activity of sponge-associated cultivable MD7_1, MD7_8, MD32_7, MD10_1 and MY11_8) endozoic fungi exhibited strong cytotoxicity (e.g.IC50<50 μg/ml) against all the four cell lines, one Penicillium raperi strain The EtOAc extracts of 12 fungi with PKS genes were sub- (MY5_3) showed strong cytotoxicity against cell line jected to screen for their cytotoxicity. As shown in Table 3,all MRC-5. Therefore, the fungi derived from South China

Aspergillus terreus MD32_7 AFK08438 Aspergillus terreus MY11_8 AFK08437 Aspergillus terreus MD11_4 AFK08435 Aspergillus terreus MD7_8 AFK08433 100 6-methylsalicylic acid synthase [Aspergillus terreus] BAA20102 Aspergillus terreus MY11_4 AFK08436 Aspergillus terreus MD10_1 AFK08442 58 Aspergillus terreus MD11_3 AFK08434 Aspergillus terreus MD7_1 AFK08441 94 putative 6-MSA-type polyketide synthase [Pertusaria erythrella] ABS11025 90 100 putative 6-MSA-type polyketide synthase [Pertusaria dactylina] ABS11024 unnamed protein product [Aspergillus oryzae RIB40] BAE65442 6-methylsalicylic acid synthase MsaS [Aspergillus clavatus] XP_001273093 56 6-methylsalicylic acid synthase [Arthroderma otae] XP_002849666 Eurotium intermedium MY21_10 AFK08444 Aspergillus ochraceus MD10_2 AFK08443 100 MSAS-type polyketide synthase [Aspergillus ochraceus] AAS98200 polyketide synthetase PksP [Neosartorya fischeri] XP_001261235 82 Penicillium raperi MY5_3 AFK08439 100 Penicillium raperi MY5_400 AFK08440 99 nonreducing polyketide synthase [Penicillium janthinellum] ADY75762 68 nonreducing polyketide synthase [Penicillium raperi] ADY75767

0.1

Fig. 2 Neighbor-joining unrooted phylogenetic tree constructed using represents 0.1 AA substitutions per site. The black cycle represents amino acid sequences of KS domain. Number at branch indicates sequence obtained in this study Bootstrap value (>50 %) from 1,000 replicates. The scale bar Fungal Diversity

Table 3 IC of 12 tested 50 μ −1 fungal extracts against human Strain Cell line and IC50 ( g·mL ) cancer cells A-549 A-375 Bel-7402 MRC-5

Aspergillus terreus MD11_3 15.52±0.99 30.74±0.95 45.31±0.98 85.6±0.98 Aspergillus terreus MD11_4 14.51±0.94 61.6±0.91 44.81±0.96 80.87±0.93 Aspergillus terreus MY11_4 19.19±0.98 56.94±0.95 53.37±0.99 160.87±0.95 Aspergillus terreus MD7_1 20.29±0.97 92.91±0.97 139.36±0.97 241.3±0.96 Aspergillus terreus MD7_8 33.19±0.98 51.25±0.95 53.18±0.98 238.31±0.99 Aspergillus terreus MD32_7 37.8±0.99 129.2±0.99 115.9±0.98 170.5±0.90 Aspergillus terreus MD10_1 13.05±0.97 30.35±0.84 21.57±0.94 46.57±0.72 Aspergillus terreus MY11_8 27.16±0.96 30.9±0.89 50.47±0.99 47.38±0.97 Penicillium raperi MY5_3 276.7±0.92 200.6±0.96 194.99±0.87 30.19±0.97 Penicillium raperi MY5_400 790.65±0.90 87.53±0.96 557.96±0.98 73.68±0.95 Aspergillus ochraceus MD10_2 483.59±0.99 109.4±0.94 65.64±0.97 162.67±0.98 Eurotium intermedium MY21_10 716.16±0.95 403.19±0.95 484.53±0.99 436.7±0.97 Cisplatin 3.70±0.96 1.11±0.95 0.55±0.99 11.58±0.90

Sea sponges are suggested to be important sources for associated fungi, the culturable fungal community con- marine drugs development. sisted of at least 14 orders: Agaricales, Agaricostilbales, Boliniales, Capnodiales, Dothideales, Eurotiales, Hypo- creales, Microascales, Mucorales, Phyllachorales, Pleo- Discussion sporales, Saccharomyceales, Wallemiales and Xylariales (Liu et al. 2010;Dingetal.2011; Zhou et al. 2011). In Fungal community associated with sponges this study, ten orders (Agaricales, Eurotiales, Hypocreales, and the association of sponge-fungi Moniliales, Pleosporales, Saccharomycetales, Sporidiobo- lales, Tremellales and Xylariales) within Ascomycota and The ecological preferences of most fungi make it probably Basidiomycota were recorded (Table 1), among which four that marine fungi are frequently associated with parasitism orders (Aphyllophorales, Moniliales, Sporidiobolales and of marine animals e.g. marine invertebrates, plants and Tremellales) were first isolated from South China Sea algae, or in benthic environments e.g. sediments. This may sponges, extending the South China Sea sponges derived in part explain why fungi have been considered to be both fungal diversity to 18 orders. In particular, four genera Bjer- nondiverse and of low abundance in upper and surface water kandera, Schizophyllum, Sporidiobolus (Basidiomycota)and column and the great diversity in marine animals and plants Yarrowia (Ascomycota) were isolated from marine sponges (Richards et al. 2012). As an important representative of for the first time (Table 4). Schizophyllum was previously marine invertebrates, sponges have been proved to harbor detected in the gene library of Suberites zeteki (Gao et al. diverse fungi by culture-dependent and culture-independent 2008), but never was isolated from sponges. Though phylum approaches (Höller et al. 2000; Gao et al. 2008; Proksch et Zygomycota, which has been isolated from sponges in other al. 2008; Wang et al. 2008; Baker et al. 2009; Li and Wang oceans (Paz et al. 2010; Menezesa et al. 2010), was not 2009; Liu et al. 2010; Menezesa et al. 2010; Paz et al. 2010; isolated from these South China Sea sponges, this study, Ding et al. 2011, Thirunavukkarasu et al. 2012; Wiese et al. together with the results from Liu et al. (2010), Ding et al. 2011;Zhouetal.2011). To our knowledge, twenty-two (2011) and Zhou et al. (2011), suggest that South China Sea orders (Boliniales, Botryosphaeriales, Capnodiales, Chaetos- sponges host diverse fungi (i.e.18 orders). phaeriales, Claromycetales, Diaporthales, Dothideales, Euro- Li and Wang (2009) classified cultivated fungal isolates tiales, Helotiales, Hypocreales, Microascales, mitosporic from sponges into 3 groups: sponge-generalists (found in all Agricomycotina, Moniliales, Mucorales, Onygenales, Phylla- sponge species), sponge-associates (found in more than one chorales, Pleosporales, Polyporales, Saccharomycetales, Sor- sponge species), and sponge-specialists (found only in one dariales, Trichosphaeriales and Xylariales) within Ascomycota sponge species). Baker et al. (2009) found that in contrast to and eight orders (Agaricales, Agaricostilbales, Corticiales, the low diversity of fungi detected by polymerase chain Malasseziales, Polyporales, Sporidiobolales, Tremellales and reaction (PCR) and reverse transcription (RT)-PCR amplifi- Wallemiales)withinBasidiomycota have been observed or cationfrom, a much higher diversity of fungi could be cul- isolated in sponges. In the case of China Sea sponge- tured. However, the revealed culturable fungal diversity is Fungal Diversity

Table 4 Summary of fungal diversity in sponges (genus level)

Fungus Sponge Reference

Ascomycota Acremonium Biemna fistulosa, Cliona viridis, Dragmacidon reticulate, Boot et al. 2006; Proksch et al. 2008; Zhang et al. 2009a; Psammocinia sp., Sigmadocia pumila, Stelletta sp., Paz et al. 2010; Wiese et al. 2011; Thirunavukkarasu Subritus carnosus, Suberites domuncula,Teichaxinella et al. 2012 sp., Tethya aurantium Alternaria Callyspongia diffusa, Cliona quadrata, Pseudosuberites Wiese et al. 2011; Thirunavukkarasu et al. 2012 andrewi, Sigmadocia pumila, Tethya aurantium Ampelomyces Gelliode fibrosa, Haliclona caerulea Li and Wang 2009 Apiospora Clathrina luteoculcitella Ding et al. 2011 Arthrinium Dragmacidon reticulata Menezesa et al. 2010 Aspergillus Amphimedon viridis, Aplysinopsis sp., Axinella damicornis, Abrell et al. 1996; Varoglu and Crews 2000; Lin et al. 2003; Biemna fistulosa, Callyspongia diffusa, Clathrina Gao et al. 2008; Proksch et al. 2008; Ein-Gil et al. 2009; luteoculcitella, Cliona quadrata, Cliona viridis, Li and Wang 2009; Lee et al. 2010; Liu et al. 2010; Dragmacidon reticulate, Fasciospongia cavernosa, Menezesa et al. 2010; Paz et al. 2010;Dingetal.2011; Gelliode fibrosa, Haliclona caerulea, Haliclona Wiese et al. 2011;Zhouetal.2011; Thirunavukkarasu madrepora, Haliclona oculata, Haliclona simulans, et al. 2012 Holoxea sp., Hyrtios proteu, Jaspis cf. coriacea, Lissodendoryx sinensis, Mycale armata, Mycale fibrexilis, Mycale laxissima, Petrosia sp., Psammocinia sp., Pseudosuberites andrewi, Sigmadocia pumila, Spongia obscura, Subritus carnosus, Suberites domuncula, Suberites zeteki, Tethya aurantium, Xestospongia exigua Ascomycete Clathrina luteoculcitella, Holoxea sp., Mycale armata, Gao et al. 2008; Ding et al. 2011; Zhou et al. 2011 Phyllospongia foliascens Aureobasidium Suberites zeteki Gao et al. 2008 Bartalinia Gelliode fibrosa Li and Wang 2009 Bionectria Amphimedon viridis, Gelliode fibrosa, Mycale laxissima, Li and Wang 2009; Menezesa et al. 2010; Paz et al. 2010 Psammocinia sp. Bipolaris Gelliode fibrosa Li and Wang 2009 Botryosphaeria Clathrina luteoculcitella, Dragmacidon reticulate Menezesa et al. 2010; Ding et al. 2011 Botrytis Tethya aurantium Wiese et al. 2011 Cephalosporium Psammocinia sp. Paz et al. 2010 Chaetomium Halichondria panacea, Ircinia oros, Ircinia variabilis, Höller et al. 2000; Proksch et al. 2008; Paz et al. 2010; Psammocinia sp., Subritus carnosus, Suberites Thirunavukkarasu et al. 2012 domuncula , Sycon sp. Candida Clathrina luteoculcitella, Haliclona caerulea, Haliclona Li and Wang 2009; Liu et al. 2010; Ding et al. 2011 simulans, Mycale armata Cladosporium Amphimedon viridis, Callyspongia aerizusa, Clathrina Jadulco et al. 2001; Gesner et al. 2005; Gao et al. 2008; luteoculcitella, Cliona viridis, Haliclona caerulea, Proksch et al. 2008; Li and Wang 2009; Liu et al. 2010; Haliclona simulans, Gelliode fibrosa, Mycale armata, Menezesa et al. 2010; Paz et al. 2010; Ding et al. 2011; Mycale laxissima, Niphates rowi, Psammocinia sp., Wiese et al. 2011; Thirunavukkarasu et al. 2012 Pseudosuberites andrewi, Suberites zeteki, Tethya aurantium, Suberites domuncula Clavicipitace Psammocinia sp. Paz et al. 2010 Clonostachys Tethya aurantium Wiese et al. 2011 Cochliobolus Amphimedon viridis, Dragmacidon reticulate, Gelliode Li and Wang 2009; Menezesa et al. 2010; Paz et al. 2010 fibrosa, Gelliode fibrosa, Mycale laxissima, Psammociniasp. Curvularia Callyspongia diffusa, Gelliode fibrosa, Niphates olemda Jadulco et al. 2002; Li and Wang 2009; Thirunavukkarasu et al. 2012 Davidiella Clathrina luteoculcitella, Holoxea sp. Ding et al. 2011 Diaporthe Gelliode fibrosa Li and Wang 2009 Didymella Haliclona caerulea, Psammocinia sp. Li and Wang 2009; Paz et al. 2010 Didymocrea Clathrina luteoculcitella Ding et al. 2011 Dothideomycetes Psammocinia sp. Paz et al. 2010 Drechslera Pseudosuberites andrewi Thirunavukkarasu et al. 2012 Emericella Callyspongia sp. cf. C. flammea, Haliclona valliculata Höller et al. 2000; Bringmann et al. 2003 Fungal Diversity

Table 4 (continued)

Fungus Sponge Reference

Emericellopsis Halichondria panacea, Myxilla incrustans, Sycon sp., Höller et al. 2000; Paz et al. 2010 Psammocinia sp. Engyodontium Suberites domuncula Proksch et al. 2008 Epicoccum Tethya aurantium Wiese et al. 2011 Eupenicillium Callyspongia sp. cf. C. flammea, Gelliode fibrosa, Höller et al. 2000; Li and Wang 2009 Haliclona caerulea, Mycale armata, Sycon sp. Eurotium Ircinia oros, Ircinia variabilis, Callyspongia diffusa, Höller et al. 2000; Wiese et al. 2011; Thirunavukkarasu Pseudosuberites andrewi, Tethya aurantium et al. 2012 Exophiala Suberites domuncula Proksch et al. 2008 Fusarium Agelas dispar, Amphimedon viridis, Biemna fistulosa, Proksch et al. 2008; Li and Wang 2009; Liu et al. 2010; Callyspongia diffusa, Clathrina luteoculcitella, Cliona Menezesa et al. 2010; Paz et al. 2010; Ding et al. 2011; viridis, Dragmacidon reticulate, Gelliode fibrosa, Wiese et al. 2011; Zhou et al. 2011; Thirunavukkarasu Haliclona simulans, Holoxea sp., Mycale laxissima, et al. 2012 Psammocinia sp., Sigmadocia pumila, Suberites domuncula, Tethya aurantium Fusicoccum Clathrina luteoculcitella, Gelliode fibrosa Li and Wang 2009; Ding et al. 2011 Gliocladium Haliclona madrepora Thirunavukkarasu et al. 2012 Gliomastix Psammocinia sp., Suberites domuncula Proksch et al. 2008; Paz et al. 2010 Glomerella Amphimedon viridis, Dragmacidon reticulate, Mycale Menezesa et al. 2010 laxissima Gymnascella Halichondria japonica Amagata et al. 2006b Gymnoascus Psammocinia sp. Paz et al. 2010 Hortaea Aplysina aerophoba, Haliclona simulans, Suberites zeteki, Brauers et al. 2001; Gao et al. 2008; Liu et al. 2010 Mycale armata, Humicola Haliclona madrepora Thirunavukkarasu et al. 2012 Hypocrea Clathrina luteoculcitella, Gelliode fibrosa, Psammocinia sp., Gao et al. 2008; Li and Wang 2009; Paz et al. 2010; Suberites zeteki Ding et al. 2011 Isaria Agelas dispar, Rhizaxinella sp. Zhou et al. 2011 Lacazia Haliclona caerulea, Mycale armata Li and Wang 2009 Lasiodiplodia Cliona viridis Thirunavukkarasu et al. 2012 Lentomitella Clathrina luteoculcitella Ding et al. 2011 Leptosphaeria Callyspongia vaginalis Höller et al. 2000 Leptosphaerulina Gelliode fibrosa Li and Wang 2009 Letendraea Acanthella cavernosa Yang et al. 2007 Metschnikowia Haliclona simulans Baker et al. 2009 Microascus Ircinia variabilis, Leucosolenia sp., Sycon sp. Höller et al. 2000 Microsphaeropsis Aplysina aerophoba Brauers et al. 2000 Monascus Ircinia variabilis Höller et al. 2000 Mycosphaerella Haliclona simulans Liu et al. 2010 Myrothecium Axinella sp., Gelliode fibrosa, Gelliode fibrosa Xie et al. 2008; Li and Wang 2009 Myxotrichum Ircinia variabilis Höller et al. 2000 Nectria Dragmacidon reticulata Menezesa et al. 2010 Niesslia Halichondria panacea, Leucosolenia sp., Myxilla Höller et al. 2000 incrustans, Sycon sp. Nigrospora Clathrina luteoculcitella, Gelliode fibrosa Li and Wang 2009; Ding et al. 2011 Nodulisporium Suberites domuncula Proksch et al. 2008 Paecilomyces Clathrina luteoculcitella, Holoxea sp., Jaspis cf. coriacea, Rahbæk et al. 1998; Proksch et al. 2008; Ding et al. 2011; Suberites domuncula, Tethya aurantium Wiese et al. 2011 Paraphaeosphaeria Gelliode fibrosa, Psammocinia sp. Li and Wang 2009; Paz et al. 2010 Phialophora Suberites domuncula Proksch et al. 2008 Penicillium Agelas clathrodes, Amphimedon viridis, Axinella sp., Edrada et al. 2002; Amagata et al. 2003; Bringmann et al. Axinella verrucosa, Callyspongia diffusa, Chondrosia 2004; Jadulco et al. 2004; Lang et al. 2007; Xin et al. reniformis, Clathrina luteoculcitella, Cliona quadrata, 2007; Fungal Diversity

Table 4 (continued)

Fungus Sponge Reference

Cliona viridis, Dragmacidon reticulate, Gelliode fibrosa, Proksch et al. 2008; Li and Wang 2009; Baker et al. Haliclona caerulea, Haliclona simulans, Holoxea sp., 2009; Gao et al. 2008; Liu et al. 2010; Menezesa et al. Lissodendoryx sinensis, Mycale armata, Mycale 2010; Paz et al. 2010; Ding et al. 2011; Wiese et al. laxissima, Mycale plumose, Pericharax heteroraphis, 2011; Zhou et al. 2011; Thirunavukkarasu et al. 2012 Petrosia ficiformis, Psammocinia sp., Pseudosuberites andrewi, Rhizaxinella sp., Suberites zeteki, Sigmadocia pumila, Suberites domuncula, Tethya aurantium, Xestospongia exigua Pestalotiopsis Clathrina luteoculcitella Ding et al. 2011 Petriella Suberites domuncula Proksch et al. 2008 Phaeosphaeria Haliclona caerulea Li and Wang 2009 Phoma Dragmacidon reticulate, Haliclona madrepora, Haliclona Proksch et al. 2008; Baker et al. 2009; Menezesa et al. simulans, Mycale laxissima, Psammocinia sp., Subritus 2010; Paz et al. 2010; Wiese et al. 2011; carnosus, Suberites domuncula, Tethya aurantium Thirunavukkarasu et al. 2012 Phomopsis Psammocinia sp. Paz et al. 2010 Plectosphaerella Agelas dispar, Psammocinia sp. Paz et al. 2010; Zhou et al. 2011 Pleosporales Psammocinia sp. Paz et al. 2010 Preussia Myxilla incrustans, Psammocinia sp. Höller et al. 2000; Paz et al. 2010 Pseudallescheria Pericharax heteroraphis Zhou et al. 2011 Pseudogymnoascus Cliona viridis, Subritus carnosus Thirunavukkarasu et al. 2012 Simplicillium Phyllospongia foliascens Zhou et al. 2011 Scopulariopsis Clathrina luteoculcitella, Suberites domuncula, Tethya Proksch et al. 2008; Ding et al. 2011; Wiese et al. 2011 aurantium Solheimia Gelliode fibrosa Li and Wang 2009 Sordariomycetes Psammocinia sp. Paz et al. 2010 Sporobolomyces Suberites domuncula Proksch et al. 2008 Sporormiella Callyspongia sp. cf. C. flammea, Cliona viridis, Subritus Höller et al. 2000 carnosus Thirunavukkarasu et al. 2012 Stachybotrys Psammocinia sp. Paz et al. 2010 Stemphylium Suberites domuncula Proksch et al. 2008 Stilbella Suberites domuncula Proksch et al. 2008 Syncephalastrum Cliona quadrata Thirunavukkarasu et al. 2012 Talaromyces Ectyplasia perox, Ircinia oros Höller et al. 2000 Thielaviopsis Fasciospongia cavernosa Thirunavukkarasu et al. 2012 Tolypocladium Suberites domuncula Proksch et al. 2008 Trichoderma Agelas dispar, Amphimedon viridis, Biemna fistulosa, Kobayashi et al. 1993; Proksch et al. 2008; Abdel-Lateffa et Cliona viridis, Dragmacidon reticulate, Gelliode fibrosa, al. 2009;LiandWang2009; Menezesa et al. 2010;Paz et al. Haliclona madrepora, Micale Cecilia, Mycale fibrexilis, 2010; Wiese et al. 2011;Zhouetal.2011; Mycale laxissima, Psammocinia sp., Sigmadocia pumila, Thirunavukkarasu et al. 2012 Suberites domuncula, Tethya aurantium Tubercularia Haliclona caerulea, Gelliode fibrosa Li and Wang 2009 Verticillium Mycale laxissima, Psammocinia sp. Menezesa et al. 2010; Paz et al. 2010 Volutella Agelas dispar, Phyllospongia foliascens, Tethya aurantium Wiese et al. 2011 Zhou et al. 2011 Xylaria Haliclona simulans Liu et al. 2010 Yarrowiaa Cinachyrella australiensis This study Basidiomycota Agaricales Amphimedon viridis Menezesa et al. 2010 Atheliales Amphimedon viridis Menezesa et al. 2010 Basidiomycete Suberites zeteki Gao et al. 2008 Bjerkanderaa Diplastrella megastellat This study Coprinellus Psammocinia sp. Paz et al. 2010 Fungal Diversity

Table 4 (continued)

Fungus Sponge Reference

Kondoa Haliclona simulans Liu et al. 2010 Malassezia Mycale armata, Suberites zeteki Gao et al. 2008 Marasmius Clathrina luteoculcitella Ding et al. 2011 Phlebia Suberites zeteki Gao et al. 2008 Polyporales Dragmacidon reticulata Menezesa et al. 2010 Schizophylluma Geodia neptuni, Suberites zeteki This study; Gao et al. 2008 Sporidiobolusa Geodia neptuni This study Sterigmatomyces Haliclona simulans Liu et al. 2010 Trichosporon Psammocinia sp. Paz et al. 2010 Tritirachium Pseudosuberites andrewi, Tethya aurantium Wiese et al. 2011; Thirunavukkarasu et al. 2012 Wallemia Haliclona simulans Liu et al. 2010 Zygomycota Rhizopus Amphimedon viridis, Dragmacidon reticulate, Psammocinia sp. Menezesa et al. 2010; Paz et al. 2010 Mucor Cliona viridis, Dragmacidon reticulate, Halichondria Mohapatra et al. 1998; Höller et al. 2000; Menezesa et al. panacea, Myxilla incrustans, Pseudosuberites andrewi, 2010; Wiese et al. 2011; Thirunavukkarasu et al. 2012 Sycon sp., Sigmadocia pumila, Spirastrella sp., Tethya aurantium Rhizomucor Clathrina luteoculcitella Ding et al. 2011 Syncephalastrum Ircinia oros, Ircinia variabilis Höller et al. 2000 a means the fungus is first isolated in this study limited greatly due to the medium and conditions used, generalists’. As shown in Table 4, most fungi in phylum which makes the comparison of fungal diversity among Basidiomycota and some fungi in phylum Ascomycota different sponges difficult. As shown in Table 4, at the genus maybe the potential sponge-specialists since they were level, the diversity of culturable fungal assemblages varied isolated only from one species sponge showing sponge greatly among different species sponges. Three fungal species-specificity. However, we still don’t know whether genera Aspergillus, Penicillium, and Eupenicillium, which the association of fungi in individual sponges is with were classified as ‘sponge-generalists’ by Li and Wang sponge specificity. At present, it is very difficult to (2009), were not ‘sponge-generalists’ according to Höller conclude which fungus belongs to the sponge-generalists, or et al. (2000), Thirunavukkarasu et al. (2012); Menezesa et associates, or specialists because of the limited sponge al. (2010). Particularly, Table 4 shows that Eupenicillium species tested and low fungal diversity observed. Mean- has been isolated in few sponges. Similarly, Fusarium and while few researches on sponge fungal communities Trichoderma, which should be sponge-generalists according using culture-independent approaches have been under- to Menezesa et al. (2010), could only be grouped as taken (Gao et al. 2008; Baker et al. 2009). The answers ‘sponge-associates’ by Höller et al. (2000) and Li and Wang may be given by the more extensive survey for more (2009), even not detected in sponges Suberites zeteki and sponges, especially the same sponge from different Mycale armata by culture-independent molecular (Gao et al. oceans, or by the newly-developed technique such as 2008). The sponge-specialist Candida in Hawaii sponge 454 pyrosequencing in the near future. Mycale armata (Li and Wang 2009) was also recovered in It is known that the majority of marine fungi belong to South China Sea sponges H. caerulea, H. simulans and C. ascomycete and basidiomycete within the Dikarya (Raghu- luteoculcitella with a relatively high frequency (Liu et al. kumar 2008; Richards et al. 2012). As one of sessile-feeding 2010; Ding et al. 2011), suggesting that it is not Mycale animals, marine sponges can filter large amounts of seawa- armata-specific. Based on the summary of sponge fungal ter every day (Fieseler et al. 2004), thus, it could be inferred diversity in Table 4,generaAspergillus (32 species that part of the fungi in sponges could come from marine sponges), Penicillium (28 species sponges), Fusarium mycoplankton by horizontal transfer. This hypothesis may (15 species sponges), Cladosporium (15 species sponges) explain the fact that Ascomycota is common in different and Trichoderma (14 species sponges) in phylum Asco- sponges to some extent. On the contrary, Gao et al. (2008) mycota, which have been isolated from sponges with observed that the profiles of fungal community varied in high frequency, are probably the candidates of ‘sponge- sponges and ambient seawater. In this study, though all the Fungal Diversity sponges were from the same location, different fungi were various fungal pigments, melanin and aflatoxin, while isolated, which suggested that these fungi were not simply the reducing PKSs are involved in the synthesis of PKS from seawater column during the filter-feeding process by compounds with various chemical reductions in struc- horizontal transfer or sea water contamination. Marine ture (Amnuaykanjanasin et al. 2005). fungal-specific clone libraries towards subsurface water col- The fungal potential for synthesizing bioactive natural umn and deep sea (500 m to 4,200 m) sediments samples products was further proved by the in vitro cytotoxicity demonstrate a simple fungal community mainly composed assay. All the 12 fungi with PKS genes displayed moderate of Dikarya (Bass et al. 2007; Edgcomb et al. 2011; to strong in vitro cytotoxic activity against human carcino- Jones 2011). The different fungal community in sponges ma cells. Particularly, 8 strains Aspergillus terreus showed from that in the sea water (Gao et al. 2008), and the highly strong cytotoxicity against multiple cell lines. This result, diverse fungi in sponges than that in the environmental sea together with the PKS detection, is in consistent with the water and sediment, suggest a complex mechanism for knowledge of Aspergillus, which has been proved to be the formation of the close association of sponge-fungi. prolific sources of biologically active secondary metabolites Maldonado et al. (2005) reported the symbiotic relation- such as polyketides with cytotoxic activity (Hendrickson et ship between and sponge, where yeast was found al. 1999; Bugni and Ireland 2004). From sponge-derived to be maternally transmitted from the soma through the Aspergillus, some novel and bioactive compounds have oocytes to the fertilized eggs. This finding proved that been isolated (Hiort et al. 2004; Ingavat et al. 2009; Liu et fungi in sponges could be captured by vertically trans- al. 2009). But, no investigation on the metabolites of fer. With the consideration of the old history of sponges sponge-derived Aspergillus terreus has been reported except and their ubiquitous distribution, it is suggested that (+)-terrain by us (Yin et al. 2012). Interestingly, though the sponges are probably important niches in fungi evolu- 8 strains Aspergillus terreus belong to the same species, they tion and radiation, for example, fungal introns were show different cytotoxicity profiles indicating that they can found to exit in some sponges aroused by horizontal gene produce different bioactive compounds. In addition, Peni- transfer (Rot et al. 2006). Despite the lack of evidence for the cillium raperi strain MY5_3 with PKS gene also showed association of sponge-fungi, a close relationship between strong cytotoxic activity against human normal embryo lung fungus and sponge including symbiosis (Maldonado et al. fibroblasts cell line MRC-5. It is known that Penicillium has 2005; Rot et al. 2006), nutrient transfer and chemical defense been widely used in the production of antibiotics e.g. pen- (e.g. anti-microorganisms) for sponges could be supposed icillin and some novel natural products have also been isolat- (Bugni and Ireland 2004;Taylor et al. 2007b; Baker et al. ed from sponge-derived Penicillium (Bringmann et al. 2004; 2009; Paz et al. 2010;Dingetal.2011). Xin et al. 2007). But, no report on the natural products of sponge-derived Penicillium raperi has been found. The The potential of culturable endozoic fungi in producing detected PKS gene and cytotoxicity activity of the 8 strains bioactive natural products of Aspergillus terreus and one strain of Penicillium raperi indicate that these South China Sea sponge-associated fungi It is suggested that sponge-associated microorganisms are are new potential sources for marine bioactive natural prod- probably involved in the production of biological natural ucts. In addition, according to this study, the integrated strat- products isolated from sponges (Müller et al. 2003;Piel egy including functional gene detection and biological activity 2004; König et al. 2006). Therefore, the fungi with bioactive analysis improves the screening efficiency of sponge- compound production potential are import resources for the associated fungi with potential for biological active natural development of marine drugs. Polyketides synthesized by products production. Of course, there are many bioactive PKS gene cluster have been isolated in sponge-derived metabolites in fungi except polyketides (e.g. peptides, alka- fungi (Kobayashi et al. 1993; Abrell et al. 1996; Rahbæk loids, terpenes, steroids, etc.) (Amagata et al. 2003, 2006a, b; et al. 1998). The result of PKS gene screening suggests the Boot et al. 2006;Saleemetal.2007; Zhang et al. 2009a; fungi associated with South China Sea sponges have the Proksch et al. 2010), thus, there is no direct proof linking the potential in synthesizing PKS compounds. Based on the presence of PKS genes to the observed cytotoxicity of the BLAST analysis, all the characterized fungal PKSs belong fungal extracts in this study. Further chemical identification to type I (multifunctional, multidomain) enzyme class. Type will be carried out to make sure the detailed bioactive natural I PKSs produce a vast array of biomedically important products derived from these fungi derived from South China secondary metabolites such as antibiotic erythromycin, Sea sponges. the immune suppressant FK506 or the antiparasitic aver- mectin derivatives (Piel et al. 2004). Type I PKSs are Acknowledgement This work was supported by the National generally divided into two subclasses: non-reducing Natural Science Foundation of China (81102417) and the High-Tech (NR) and reducing. NR-PKSs include those synthesizing Research and Development Program of China (2011AA09070203) Fungal Diversity

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