In most studies on fungi associated with sponges the taxonomic classification of the fungi was based exclusively on morphological characteristics and in many cases identification was possible only at the genus level [17]. This can be attributed to the fact that taxonomic identification of fungi at the species level is not always easy. It is impaired by the fact that under laboratory conditions many fungi do not express reproductive features like conidia or ascomata, which represent important traits for identification. These fungi are classified as “mycelia sterilia”.

Therefore, morphological criteria as well as sequence information and the determination of phylogenetic relationships are considered to be necessary for the identification of fungi. Consequently, we have combined the morphological characterization with a PCR-based analysis using ITS1-5.8S-rRNA-ITS2 gene sequences to identify 81 fungi isolated from Tethya aurantium. Based on these criteria the strains could be identified to the species level (Figure 1, Table 1).

First of all, morphological criteria enabled the identification of most of the fungal isolates at the genus level (Table 1). However, under the culture conditions applied, 11 strains did not produce spores and were designed as “mycelia sterilia”. A morphological classification of these strains was not possible. Despite the formation of spores, another four strains could not be classified on the basis of morphological characteristics.

In order to verify the results of the morphological examination and identify the strains at the species level, they were subjected to ITS1-5.8S-ITS2 gene sequence analysis. The results obtained from this sequence analysis corresponded well with those from the morphological identification (Table 1, Figure 2) and in addition allowed identification of those strains not identified microscopically. In most cases, the sequence data and the phylogenetic relationships allowed the identification at the species level. Results from BLAST search are depicted in Table 1. Taking together morphological and genetic characteristics, most isolates belong to the Ascomycotina with representatives of the fungal classes Dothideomycetes (24 isolates), Eurotiomycetes, (25 isolates), Sordariomycetes (30 isolates) and Leotiomycetes (1 isolate). Only a single isolate (strain LF538) was classified as belonging to the Mucoromycotina.

As shown in the phylogenetic tree (Figure 2), 10 isolates of the Dothideomycetes closely affiliated to fungal species of the order Capnodiales (Cladosporium spp. and its teleomorph Davidiella). Within the order Pleosporales (13 isolates) 5 isolates were closely related to Alternaria sp., including Lewia infectoria as teleomorph. From the same order we also isolated 2 strains of the species Epicoccum nigrum and 1 Paraphaeosphaeria strain. Furthermore, 5 isolates of the genus Phoma were found, of which one isolate (strain LF258) shows only 91% sequence similarity to Septoria arundinacea as next relative according to BLAST results (Table 1) and presumably represents a new species in the Phoma lineage. The low similarity of this isolate to known sequences is also reflected in the position in the phylogenetic tree, clustering distinct from other Phoma species. A single isolate (strain LF241) was assigned to Sphaeropsis sapinea/Botryosphaeria sp. within the order Botryosphaeriales in the class Dothideomycetes.

All 25 isolates assigned to the class Eurotiomycetes were affiliated to the order Eurotiales and are represented by 14 isolates closely affiliating to Penicillium species (P. glabrum, P. virgatum/ P. brevicompactum, P. griseofulvum/P. commune, P. chrysogenum, P. sclerotiorum/P. citreonigrum, P. citreoviride/P. roseopurpureum, P. canescens), 10 representatives of the genus Aspergillus (including 2 of its teleomorphs, Petromyces alliaceus and Eurotium chevalieri) and 1 isolate of Paecilomyces.

The 30 isolates affiliated to the class Sordariomycetes were grouped into the orders Hypocreales (26 isolates), Microascales (2 isolates) and Xylariales (1 isolate). Within the Hypocreales, one isolate each was closely related to Beauveria bassiana (strain LF256), Tritirachium album (strain LF562), Verticillium sp. (strain LF496), and Volutella ciliata (strain LF246). The most frequent genera within this order were Fusarium (13 isolates), Trichoderma (5 isolates, including Hypocrea teleomorphs) and Clonostachys (4 isolates, including teleomorphs of Bionectria). The Microascales were represented by two isolates of the genus Scopulariopsis. One of these (strain LF064), morphologically classified as Scopulariopsis murina, was distantly related to the next cultured relative (89% according to BLAST search) and appears as a sister group to the Scopulariopsis lineage in the phylogenetic tree. The order Xylariales was represented by only a single isolate (strain LF550), Bartalinia robillardoides. As the only representatives of the order Helotiales within the class Leotiomycetes strain LF248 was closely related to Botryotinia fuckeliana.

The combination of microscopic and genetic analyses has proven to be a reliable method for the identification of fungal isolates and results from both approaches corresponded quite well (Table 1, Figure 2). The reliable identification of isolates is a fundamental prerequisite in order to characterize the producers of marine natural products [3], to determine the occurrence of fungal species in different habitats, and to correlate distinct secondary metabolite patterns to fungal species. Therefore, we highly recommend that morphological identification of fungal isolates consequently should be verified by molecular means and thus raise the number of reliably identified species in public databases.

A number of investigations, mainly with the aim of finding novel natural products, showed that most marine sponges harbor a plethora of cultivable fungi within their tissue [10–15,18]. Despite the large number of fungi isolated from sponges, a selective accumulation of specific taxa within sponges and a truly marine nature of these fungi is doubted, which is why they are commonly referred to as “marine-derived” [27,28]. In fact, it could be shown that the phylogenetic diversity of fungi isolated from different sponges varies [17]. It has also been stated that the taxa frequently isolated from sponges resemble those described from terrestrial habitats [11,16,28]. This is in good accordance with our results, showing representatives of Acremonium, Aspergillus, Fusarium, Penicillium, Phoma, and Trichoderma to be abundant in Tethya aurantium and with those of Wang (2006) demonstrating that they also are widely distributed among different sponges from various locations [29]. Although it appears that the marine environment indeed provides various habitats for fungi, this has rarely been demonstrated. Nonetheless, due to the accumulation of fungi within sponges a large number of strains can be isolated which increases the probability to find representatives of less common taxa which might produce unprecedented secondary metabolites. For example, fungi belonging to the genera Beauveria, Botryosphaeria, Epicoccum, Tritirachium, and Paraphaeosphaeria have rarely been obtained from marine sponges [11,18,24] and, to the best of our knowledge, we have isolated Bartalinia sp. and Volutella sp. from a marine sponge for the first time.

Although evidence is presented that some bacterial symbionts of sponges are the producers of metabolites originally assumed to be produced by the sponge [4], equivalent evidence for fungi is lacking. There is actually little evidence for sponge-specific fungal associations and the only reports on this matter deals with the above mentioned Koralionastes species and a yeast living in symbiosis with the sponge Chondrilla sp. [30]. In fact, most of the studies had the biotechnological potential of sponge-derived fungi in mind but not the ecological role. Our culture-dependent approach was not considered to approach the aspect of specificity of the association with the sponge and molecular-based studies would be more suited to identify specifically sponge-associated fungi.

With the cultivation-based approach used in this study, we obtained a variety of strains from a surprisingly broad range of phylogenetic groups of fungi. A selection of the isolated and identified fungi was subjected to analysis of their secondary metabolite profiles. Strains were selected in order to represent a wide spectrum of genera, representatives of a variety of different genera, some known to include strains and species known for the production of secondary metabolites others from less common taxa. Some of the strains selected according to systematic criteria did not produce detectable amounts of secondary metabolites under the applied culture conditions. Unraveling their potential of secondary metabolite production will require more intense studies. Those extracts which did contain at least one compound in significant amounts were analyzed by HPLC with DAD (UV)- and MS-detection and the metabolites which could be identified are listed in Table 2. A high percentage of the substances identified this way could be verified by ¹H NMR spectroscopy (see Table 2). The majority of these metabolites have been reported from fungi before. Two of our previous reports on metabolites from fungi isolated from Tethya aurantium deal with the antiproliferative scopularides A–B [25,26] and the sorbifuranones A–C [31].

For four compounds, database searches [33,42–43] did not lead to a hit (B, C, D) or no publication was available (A). The structure elucidations of compounds A and B, metabolites with modified diketopiperazine substructures, and compound D, a new azaphilone derivative, are in progress. Compound C was identified as the new metabolite cillifuranone and its structure is described in the following.

Penicillium strain LF066, the producer of cillifuranone, was singled out for further investigations, because first surveys proved it to be a very potent producer of secondary metabolites. From the same strain, sorbifuranone B and C as well as 2′,3′-dihydrosorbicillin have already been described by Bringmann et al. [31] and it was obvious that the full potential of the strain had not been exploited, yet. Further analysis led to the detection of xanthocillines and sorbifuranones and the isolation of sorbifuranone B, meleagrin, roquefortin C, a couple of ergochromes as well as the new cillifuranone (1) whose structure was elucidated based on 1D and 2D NMR experiments.

The ¹³C NMR spectrum of 1 displayed 10 clearly distinguishable carbon signals which was in good agreement with the molecular formula C₁₀H₁₂O₄, deduced from the result of a HRESI-MS measurement (calculated for C₁₀H₁₂O₄Na 219.0628, measured 219.0627). The carbon signals included resonances belonging to three carbonyl or enol carbons (δ_C 170.9, 196.7 and 201.7), three sp³ hybridized methylene carbons (δ_C 20.9, 31.6 and 76.3), one methyl group (δ_C 14.0), two olefinic methines (δ_C 118.5 and 133.0) and finally one quaternary olefinic carbon (δ_C 112.6). The structure of the molecule could be delineated from 1D (¹H, ¹³C and DEPT) and 2D NMR (¹H-¹³C HSQC, ¹H-¹H COSY and ¹H-¹³C HMBC) spectra. From the ¹H-¹H COSY spectrum two separate spin systems could be identified. The first one consisted of the olefinic methine groups CH-9 (δ_C 133.0, δ_H 7.32) and CH-10 (δ_C 118.5, δ_H 6.83), forming an E-configured double bond as proven by their ³J coupling constant of 16 Hz. The corresponding protons H-9 and H-10 both showed ¹H-¹³C HMBC correlations to the carboxyl carbon C-11 (δ_C 170.9) as well as to the quarternary carbons of the furanone ring, C-3 (δ_C 201.7) to C-5 (δ_C 196.7). C-3 was the ketone carbonyl group included in the furanone substructure which was in accordance with its chemical shift. C-4 (δ_C 112.6) and C-5 were also part of the furanone and formed a tetrasubstituted double bond in which C-5 was located adjacent to an oxygen atom. Compared to an unsubstituted enol, the resonance of C-5 was shifted further downfield due to the conjugation of the double bond Δ^4,5 with the carbonyl carbon C-3. Apart from C-9 of the double bond Δ^9,10, the carbonyl carbon C-3 and the oxygen atom of the furanone ring, Δ^4,5 was also connected to C-6 of the second spin system consisting of the methylene groups CH₂-6 (δ_C 31.6, δ_H 2.76) and CH₂-7 (δ_C 20.9, δ_H 1.76) as well as the methyl-group CH₃-8 (δ_C 14.0, δ_H 1.03). Thus, the second spin system evidently was an n-propyl-chain. The furanone ring was completed with the methylene group CH₂-2 (δ_C 76.3, δ_H 4.67). Its ¹H and ¹³C shifts proved it to be linked to an oxyen atom, the ¹H-¹³C HMBC correlations to C-3 and C-5 secured its exact position. Thus, the structure of cillifuranone (1) could be unambiguously determined (Figure 3, Table 3).

Cillifuranone (1) was tested in a panel of bioassays evaluating the compound with respect to cytotoxic, antimicrobial and enzyme inhibitory activity. Very low activity was only found against Xanthomonas campestris (24% growth inhibition) and Septoria tritici (20% growth inhibition) at a concentration of 100 μM.

Strain LF066 was identified as Penicillium chrysogenum, a species that in our experience often produces metabolites deriving from sorbicillinol as a biosynthetic precursor (sorbicillinoids). The detection of bisvertinolone and the sorbifuranones in culture extracts of the fungus was consistent with this experience. Furanone substructures are abundant in natural products and can be found in metabolites from bacteria, fungi and plants [42] and presumably are products from different biosynthetic pathways [44–46]. From the genus Penicillium a number of furanone containing compounds has been described, including simple small molecules like penicillic acid, but also more complex ring structures such as the rotiorinols [47] or rugulovasines [48]. In most cases the furanone ring is a furan-2(5H)-one, whereas in cillifuranone (1) we have a furan-3(2H)-one. With that differentiation being made the number of related compounds gets fewer, furan-3(2H)-ones do not seem to be as ubiquituous as the furan-2(5H)-ones. From Penicillium strains, apart from the above mentioned sorbifuranones, berkeleyamide D [49] and trachyspic acid [50], both spiro compounds like sorbifuranone C, are examples. The new cillifuranone represents a substructure of the sorbifuranones, albeit with a different configuration of the exocyclic double bond, and represents the substructure which makes the sorbifuranones unique in the compound class of the sorbicillinoids. Just recently Bringmann et al. [31] published the structures of the sorbifuranones and postulated 1 to be an intermediate in their biosynthesis (Figure 4) which makes the isolation of 1 a very interesting result. The only difference between the postulated intermediate and our structure is, as stated above, the configuration of the double bond. However, in the crude extract of the strain, we detected two isomers with the same molecular weight and a very similar UV-spectrum, so that we assume that both isomers were present, but after the isolation process only the E-isomer was obtained, so that it might be the favoured configuration under the applied conditions.

The Limsky kanal (Canal di Lemme or Limsky channel) is a semi-closed fjord-like bay in the Adriatic Sea nearby Rovinj (Istrian Peninsula, Croatia). It is situated along an east-west axis, with an approximate length of 1 km and a maximum width of about 650 m and reaches a maximum depth of 32 m [51]. The sampling site was located at N45°7972′ and E13°43,734′.

Several specimens (13) of the Mediterranean sponge Tethya aurantium were collected by scuba diving. They were obtained in April 2003, June 2004, May 2005 and August 2006 in a depth of 5–15 m. The sponges were collected into sterile plastic bags, cooled on ice and transported immediately to the laboratory, where they were washed three times with sterile filtered seawater (0.2 μm). The sponge tissue was then cut into small pieces of approximately 0.1 cm³ each, which were either placed directly onto GPY agar plates (LF236 to LF255) or homogenized and diluted with membrane-filtered seawater (all other isolates).

Fungi were isolated on a low nutrient GPY agar, based on natural seawater of 30 PSU, containing 0.1% glucose, 0.05% peptone, 0.01% yeast extract and 1.5% agar. Small pieces of sponge tissue or 50 μL of the homogenate (undiluted or 1:10 or 1:100 diluted with sterile seawater) were used as inoculum. The agar plates were incubated for periods of 3 days to 4 weeks and were checked regularly for fungal colonies, which were then transferred to GPY agar plates. Pure cultures were used for morphological identification by light microscopy and for scanning electron microscopy. Fungal isolates were stored as agar slant cultures at 5 °C, and additionally were conserved at −80 °C using Cryobank vials (Mast Diagnostica).

The morphology of GPY agar grown fungal isolates was studied using a stereo microscope (10–80× magnification) and a phase-contrast microscope (300–500× magnification). By this method, the majority of spore-producing isolates could be identified up to the generic level using the tables of Barnett and Hunter [52] and, for more detailed descriptions, the site of MycoBank [53]. Morphological identification of the selected pure cultures was supported by light microscopy and scanning electron microscopy.

For electron microscopy, young GPY agar colonies were cut in 1 cm² samples, transferred through an ethanol series (30, 50, 70, 90, 3 × 100%; each 15 min) and subsequently critical-point-dried in liquid carbon dioxide (Balzers CPD030). Samples were sputter-coated with gold-palladium (Balzers SCD004) and analyzed with a ZEISS DSM 940 scanning electron microscope.

DNA-extraction was performed using the Precellys 24 system (Bertin Technologies). To one vial of a Precellys grinding kit with a glass beads matrix (diameter 0.5 mm, peqlab Biotechnologies GmbH) 400 μL DNAse-free water (Fluka) were added. Cell material from the fungal culture was then transferred to this vial and homogenized two times for 45 s at a shaker frequency of 6500. The suspension was centrifuged at 6000 g for 10 min and 15 °C. The supernatant was stored at −20 °C until further use in PCR.

Fungal specific PCR by amplifying the ITS1-5.8S rRNA-ITS2 fragment was carried out using puReTaq™ Ready-To-Go™ PCR Beads (GE HEalthcare) with the ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) primers according to White et al. [54]. PCR was conducted as follows: initial denaturation (2 min at 94 °C), 30 cycles of primer denaturation (40 s at 94 °C), annealing (40 s at 55 °C), and elongation (1 min at 72 °C) followed by a final elongation step (10 min at 72 °C). PCR products were checked for correct length (complete ITS1, 5.8S rRNA and ITS2 fragment length of Penicillium brevicompactum strain SCCM 10-I3 (EMBL-acc. No. EU587339 is 494 nucleotides), a 1% agarose gel in 1× TBE buffer (8.9 mM Tris, 8.9 mM borate, 0.2 mM EDTA).

PCR products were sequenced using the ABI PRISM^® BigDye™ Terminator Ready Reaction Kit (Applied Biosystems) on an ABI PRISM^® 310 Genetic Analyzer (Perkin Elmer Applied Biosystems). The ITS1 primer was used for sequencing. Sequence data were edited with ChromasPro Version 1.15 (Technelysium Pty Ltd.). Sequences from fungal strains obtained during this study were submitted to the EMBL database and were assigned accession numbers (FR822769-FR822849). Closest relatives were identified by sequence comparison with the NCBI Genbank database using BLAST (Basic Local Alignment Search Tool) [55]. Sequences were aligned using the ClustalX version 2.0 software [56] and the alignment was refined manually using BioEdit (version 7.0.9.0) [57]. For alignment construction, ITS1-5.8S-ITS2 gene fragments from closest cultured relatives according to BLAST as well as type strains were used, whenever possible. However, not from all fungal species, ITS sequences from type strains were available in NCBI/Genbank. The ITS1-5.8S-ITS2 gene sequence of Saccharomyces boulardii strain UOA/HCPF EM10049 (acc. No. FJ433878) was used as outgroup sequence for phylogenetic calculations. Phylogenetic calculations were performed with all closest relatives according to BLAST results (data not shown). For clarity, not all of these sequences were included in Figure 2. Phylogeny was inferred using MrBayes version 3.1 [58,59], assuming the GTR (general time reversible) phylogenetic model with 6 substitution rate parameters, a gamma-shaped rate variation with a proportion of invariable sites and default priors of the program. 1,000,000 generations were calculated and sampled every 1000th generation. Burn-in frequency was set to 25% of the samples. The consensus tree was edited in Treeview 1.3 [60].

The fungi were inoculated in 2 L Erlenmeyer flasks containing 750 mL modified Wickerham-medium [61], which consisted of 1% glucose, 0.5% peptone, 0.3% yeast extract, 0.3% malt extract, 3% sodium chloride (pH = 6.8). After incubation for 11–20 days at 28 °C in the dark as static cultures, extracts of the fungi were obtained. The mycelium was separated from the culture medium and extracted with ethanol (150 mL). The fermentation broth was extracted with ethyl acetate (400 mL). Both extracts were combined. Alternatively, cells and mycelium were not separated and the culture broth was extracted together with the cells of some cultures using ethyl acetate. After evaporation of the solvents, the powdery residue was reextracted with ethyl acetate (100 mL). The resulting residues were dissolved in 20 mL methanol and subjected to analytical HPLC-DAD-MS.

UV-spectra of the identified metabolites were obtained on a NanoVue (GE Healthcare). NMR spectra were recorded on a Bruker DRX500 spectrometer (500 and 125 MHz for ¹H and ¹³C NMR, respectively), using the signals of the residual solvent protons and the solvent carbons as internal references (δ_H 3.31 ppm and δ_C 49.0 ppm for methanol-d4). High-resolution mass spectra were acquired on a benchtop time-of-flight spectrometer (MicrOTOF, Bruker Daltonics) with positive electrospray ionization. Analytical reversed phase HPLC-DAD-MS experiments were performed using a C₁₈ column (Phenomenex Onyx Monolithic C18, 100 × 3.00 mm) applying an H₂O (A)/MeCN (B) gradient with 0.1% HCOOH added to both solvents (gradient: 0 min 5% B, 4 min 60% B, 6 min 100% B; flow 2 mL/min) on a VWR Hitachi Elite LaChrom system coupled to an ESI-ion trap detector (Esquire 4000, Bruker Daltonics).

Preparative HPLC was carried out using a VWR system consisting of a P110 pump, a P311 UV detector, a smartline 3900 autosampler and a Phenomenex Gemini-NX C18 110A, 100 × 50 mm, column or a Merck Hitachi system consisting of an L-7150 pump, an L-2200 autosampler and an L-2450 diode array detector and a Phenomenex Gemini C18 110A AXIA, 100 × 21.20 mm, column.

For the preparation of cillifuranone (1), the same solvents were used as for the analytical HPLC, with a gradient from 10% B, increasing to 60% B in 17 min, to 100% B from 17 to 22 min. 1 eluted with a retention time of 6.8 min and the amount of 23.2 mg of 1 could be obtained from a culture volume of 10 L.

Properties of cillifuranone (1): pale yellow needles; UV (MeOH) λ_max (logɛ) 278 (4.36); for 1D and 2D NMR data see Table 3 and SI; HRESIMS m/z 219.0627 (calcd for C₁₀H₁₂O₄Na 219.0628).

Possible antimicrobial effects of cillifuranone (100 μM) were tested against Bacillus subtilis (DSM 347), Brevibacterium epidermidis (DSM 20660), Dermabacter hominis (DSM 7083), Erwinia amylovora (DSM 50901), Escherichia coli K12 (DSM 498), Pseudomonas fluorescens (NCIMB 10586), Propionibacterium acnes (DSM 1897^T), Pseudomonas aeruginosa (DSM 50071), Pseudomonas syringae pv. aptata (DSM 50252), Staphylococcus epidermidis (DSM 20044), Staphylococcus lentus (DSM 6672), Xanthomonas campestris (DSM 2405), Candida albicans (DSM 1386), Phytophthora infestans, and Septoria tritici according to Schneemann et al. [62]. In addition, cytotoxic activity of cillifuranone (50 μM) towards the human hepatocellular carcinoma cell line HepG2 and the human colon adenocarcinom cell line HT29 were performed according to Schneemann et al. [62]. Inhibitory activities of cillifuranone (10 µM) against the enzymes acetylcholinesterase, phosphodiesterase (PDE-4B2), glycogen synthase kinase 3β, protein tyrosin phosphatase 1B, and HIV-1 reverse transcriptase were tested according to Helaly et al. [63].