, Volume 66, Issue 2, pp 99–106 | Cite as

Diversity of fungi isolated from three temperate ascidians

  • Susanna López-LegentilEmail author
  • Patrick M. Erwin
  • Marta Turon
  • Oded Yarden
Short Communication


Ascidians are known to harbor diverse and host-specific bacterial and archaeal communities in their tunic. However, to date, only one ascidian species has been investigated to assess symbiotic relationships with fungi and the extent of their diversity. In this study, we isolated and identified 37 strains of fungi in association with three common ascidian species in the NW Mediterranean Sea: Cystodytes dellechiajei, Didemnum fulgens, and Pycnoclavella communis, and 15 additional strains from concentrated seawater samples collected around the animals. Most of the isolated fungi were classified within four orders: Eurotiales (predominantly Penicillium spp.), Pleosporales, Hypocreales (predominantly Trichoderma spp.), and Capnodiales (Cladosporium spp.). Three additional fungal isolates from C. dellechiajei and D. fulgens belonged to the orders Helotiales, Phylachorales and Microascales, and matched to well-known plant and human pathogens (Botrytis cinerea, Plectosphaerella cucumerina and Scopulariopsis brevicaulis). Host-specificity of ascidian-associated fungi was not apparent and thus the significance of ascidian-fungal associations for ascidian wellbeing and their possible ecological roles remain unknown.


Fungus Mediterranean Sea ITS Sea-squirt Tunicata Pathogens 



This research was funded by the Spanish Government project MARSYMBIOMICS CTM2013-43287-P, and the Catalan Government grant 2014SGR-336 for Consolidated Research Groups.


  1. Ainsworth TD, Thurber RV, Gates RD (2010) The future of coral reefs: a microbial perspective. Trends Ecol Evol 25:233–240CrossRefPubMedGoogle Scholar
  2. Ainsworth TD, Krause L, Bridge T, Torda G, Raina J-B, Zakrzewski M, Gates RD, Padilla-Gamino JL, Spalding HL, Smith C, Woolsey ES, Bourne DG, Bongaerts P, Hoegh-Guldberg O, Leggat W (2015) The coral core microbiome identifies rare bacterial taxa as ubiquitous endosymbionts. ISME J in pressGoogle Scholar
  3. Alker AP, Smith GW, Kim K (2001) Characterization of Aspergillus sydowii (Thom et Church), a fungal pathogen of Caribbean sea fan corals. Hydrobiologia 460:105–111CrossRefGoogle Scholar
  4. Amend AS, Barshis DJ, Oliver TA (2012) Coral-associated marine fungi form novel lineages and heterogenous assemblages. ISME J 6:1291–1301PubMedCentralCrossRefPubMedGoogle Scholar
  5. Appleton DR, Copp BR (2003) Kottamide E, the first example of a natural product bearing the amino acid 4-amino-1, 2-dithiolane-4-carboxylic acid (Adt). Tetrahedron Lett 44:8963–8965CrossRefGoogle Scholar
  6. Appleton DR, Page MJ, Lambert G, Berridge MV, Copp BR (2002) Kottamides A–D: novel bioactive imidazolone-containing alkaloids from the New Zea- land ascidian Pycnoclavella kottae. J Org Chem 67:5402–5404CrossRefPubMedGoogle Scholar
  7. Blunt JW, Copp BR, Keyzers RA, Munro MHG, Prinsep MR (2014) Marine natural products. Nat Prod Rep 31:160–258CrossRefPubMedGoogle Scholar
  8. Blunt JW, Copp BR, Keyzers RA, Munro MHG, Prinsep MR (2015) Marine natural products. Nat Prod Rep 32:116–211CrossRefPubMedGoogle Scholar
  9. Bontemps N, Bry D, López-Legentil S, Simon-Levert A, Long C, Banaigs B (2010) Structures and antimicrobial activities of pyridoacridine alkaloids isolated from different chromotypes of the ascidian Cystodytes dellechiajei. J Nat Prod 73:1044–1048CrossRefPubMedGoogle Scholar
  10. Bourne DG, Garren M, Work TM, Rosenberg E, Smith GW, Harvell CD (2009) Microbial disease and the coral holobiont. Trends Microbiol 17:554–562CrossRefPubMedGoogle Scholar
  11. Bry D, Banaigs B, Long C, Bontemps N (2011) New pyridoacridine alkaloids from the purple morph of the ascidian Cystodytes dellechiajei. Tetrahedron Lett 52:3041–3044CrossRefGoogle Scholar
  12. Carlucci A, Raimondo ML, Santos JC, Phillips AJL (2012) Plectosphaerella species associated with root and collar rots of horticultural crops in southern Italy. Persoonia 28:34–48PubMedCentralCrossRefPubMedGoogle Scholar
  13. Carman MR, Morris JA, Karney RC, Grunden DW (2010) An initial assessment of native and invasive tunicates in shellfish aquaculture of the North American east coast. J Appl Ichthyol 26:8–11CrossRefGoogle Scholar
  14. Carr G, Tay W, Bottriell H, Andersen SK, Mauk AG, Andersen RJ (2009) Plectosphaeroic acids A, B, and C, Indoleamine 2,3-Dioxygenase inhibitors produced in culture by a marine isolate of the fungus Plectosphaerella cucumerina. Org Lett 11:2996–2999CrossRefPubMedGoogle Scholar
  15. Cuenca-Estrella M, Gomez-Lopez A, Mellado E, Buitrago MJ, Monzón A, Rodriguez-Tudela JL (2003) Scopulariopsis brevicaulis, a fungal pathogen resistant to broad-spectrum antifungal agents. Antimicrob Agents Chemother 47:2339–2341PubMedCentralCrossRefPubMedGoogle Scholar
  16. Dhar J, Carey PB (1993) Scopulariopsis brevicaulis skin lesions in an AIDS patient. AIDS 7:1283–1284CrossRefPubMedGoogle Scholar
  17. Easson CG, Thacker RW (2014) Phylogenetic signal in the community structure of host-specific microbiomes of tropical marine sponges. Front Microbiol 5:Article 532CrossRefPubMedGoogle Scholar
  18. Ein-Gil N, Ilan M, Carmeli S, Smith GW, Pawlik JR, Yarden O (2009) Presence of Aspergillus sydowii, a pathogen of gorgonian sea fans in the marine sponge Spongia obscura. ISME J 3:752–755CrossRefPubMedGoogle Scholar
  19. Elad Y, Williamson B, Tudzynski P, Delen N (2007) Botrytis spp. and diseases they cause in agricultural systems—an Introduction. In: Elad Y, Williamson B, Tudzynski P, Delen N (eds) Botrytis: biology, pathology and control. Springer, The NetherlandsCrossRefGoogle Scholar
  20. Erwin PM, López-Legentil S, Schuhmann PW (2010) The pharmaceutical value of marine biodiversity for anti-cancer drug discovery. Ecol Econ 70:445–451CrossRefGoogle Scholar
  21. Erwin PM, Pita L, López-Legentil S, Turon X (2012) Stability of sponge-associated bacteria over large seasonal shifts in temperature and irradiance. Appl Environ Microbiol 78:7358–7360PubMedCentralCrossRefPubMedGoogle Scholar
  22. Erwin PM, Pineda MC, Webster N, Turon X, López-Legentil S (2013) Small core communities and high variability in bacteria associated with the introduced ascidian Styela plicata. Symbiosis 59:35–46CrossRefGoogle Scholar
  23. Erwin PM, Pineda MC, Webster NS, Turon X, López-Legentil S (2014) Down under the tunic: bacterial biodiversity hotspots and widespread ammonia-oxidizing archaea in coral reef ascidians. ISME J 8:575–588PubMedCentralCrossRefPubMedGoogle Scholar
  24. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791CrossRefGoogle Scholar
  25. Geiser DM, Taylor JW, Ritchie KB, Smith GW (1998) Cause of sea fan death in the West Indies. Nature 394:137–138CrossRefGoogle Scholar
  26. Höller U, Wright AD, Matthée GF, Konig GM, Draeger S, Aust H-J, Schulz B (2000) Fungi from marine sponges: diversity, biological activity and secondary metabolites. Mycol Res 104:1354–1365CrossRefGoogle Scholar
  27. Imhoff JF, Kajahn I, Lang G, Wiese J, Peters A (2010) Production and use of antimumoral, antibiotic and insecticidal cyclodepsipeptides. Patent WO 2010/142258Google Scholar
  28. Jiao J-Y, Zhu T-J, Zhu W-M, Du L, Wang C-Y, Guan H-S, Gu QQ (2009) Isolation of sponge-associated fungi and screening of their antitumor activity. J Ocean Univ China 39:42–46Google Scholar
  29. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Mentjies P, Drummond AJ (2012) Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–1649PubMedCentralCrossRefPubMedGoogle Scholar
  30. Koch L, Lodin A, Herold I, Ilan M, Carmeli S, Yarden O (2014) Sensitivity of Neurospora crassa to a marine-derived Aspergillus tubingensis anhydride exhibiting antifungal activity that is mediated by the MAS1 protein. Marine Drugs 12:4713–4731PubMedCentralCrossRefPubMedGoogle Scholar
  31. Lambert G (2001) A global overview of ascidian introductions and their possible impact on the endemic fauna. In: Sawada H, Tokosawa H, Lambert CC (eds) The biology of ascidians. Springer, Tokyo, JapanGoogle Scholar
  32. Lambert G (2005) Ecology and natural history of the protochordates. Can J Zool 83:34–50CrossRefGoogle Scholar
  33. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) ClustalW and ClustalX version 2. Bioinformatics 23:2947–2948CrossRefPubMedGoogle Scholar
  34. Li Q, Wang G (2009) Diversity of fungal isolates from three Hawaiian marine sponges. Microbiol Res 164:233–241CrossRefPubMedGoogle Scholar
  35. López-Legentil S, Turon X (2005) How do morphotypes and chemotypes relate to genotypes? The colonial ascidian Cystodytes (Polycitoridae). Zool Scr 34:3–14CrossRefGoogle Scholar
  36. López-Legentil S, Dieckmann R, Bontemps-Subielos N, Turon X, Banaigs B (2005) Qualitative variation of alkaloids in color morphs of Cystodytes (Ascidiacea). Biochem Syst Ecol 33:1107–1119CrossRefGoogle Scholar
  37. López-Legentil S, Bontemps-Subielos N, Turon X, Banaigs B (2006a) Temporal variation in the production of four secondary metabolites in a colonial ascidian. J Chem Ecol 32:2079–2084CrossRefPubMedGoogle Scholar
  38. López-Legentil S, Turon X, Schupp P (2006b) Chemical and physical defenses against predators in Cystodytes (Ascidiacea). J Exp Mar Biol Ecol 332:27–36CrossRefGoogle Scholar
  39. López-Legentil S, Erwin PM, Velasco M, Turon X (2013) Growing or reproducing in a temperate sea: optimization of resource allocation in a colonial ascidian. Invertebr Biol 132:69–80CrossRefGoogle Scholar
  40. López-Legentil S, Turon X, Espluga R, Erwin PM (2015) Temporal stability of bacterial symbionts in a temperate ascidian. Front Microbiol. doi: 10.3389/fmicb.2015.01022
  41. Martínez-García M, Díaz-Valdés M, Ramos-Esplà A, Salvador N, Lopez P, Larriba E, Antón J (2007a) Cytotoxicity of the ascidian Cystodytes dellechiajei against tumor cells and study of the involvement of associated microbiota in the production of cytotoxic compounds. Marine Drugs 5:52–70PubMedCentralCrossRefPubMedGoogle Scholar
  42. Martínez-García M, Díaz-Valdés M, Wanner G, Ramos-Esplà A, Antón J (2007b) Microbial community associated with the colonial ascidian Cystodytes dellechiajei. Environ Microbiol 9:521–534CrossRefPubMedGoogle Scholar
  43. Martínez-García M, Koblízek M, López-Legentil S, Antón J (2011) Epibiosis of oxygenic phototrophs containing chlorophylls a, b, c, and d on the colonial ascidian Cystodytes dellechiajei. Microb Ecol 61:13–19CrossRefPubMedGoogle Scholar
  44. Menezes CB, Bonugli-Santosa RC, Miquelettoa PB, Passarinia MR, Silvaa CH, Justoa MR, Leala RR, Fantinatti-Garbogginia F, Oliveiraa VM, Berlinckb RG, Sette LD (2010) Microbial diversity associated with algae, ascidians and sponges from the north coast of São Paulo state, Brazil. Microbiol Res 165:466–482CrossRefPubMedGoogle Scholar
  45. Montenegro TGC, Rodrigues FAR, Jimenez PC, Angelim AL, Melo VMM, Rodrigues Filho E, de Oliveira MCF, Costa-Lotufo LV (2012) Cytotoxic activity of fungal strains isolated from the ascidian Eudistoma vannamei. Chem Biodivers 9:2203–2209CrossRefPubMedGoogle Scholar
  46. Morrison-Gardiner S (2002) Dominant fungi from Australia coral reefs. Fungal Divers 9:105–121Google Scholar
  47. Mouchka ME, Hewson I, Harvell CD (2010) Coral-associated bacterial asemblages: current knowledge and the potential for climate-driven impacts. Integr Comp Biol 50:662–674CrossRefPubMedGoogle Scholar
  48. Park MS, Fong JJ, Oh S-Y, Kwon KK, Sohn JH, Lim YW (2014) Marine-derived Penicillium in Korea: diversity, enzyme activity, and antifungal properties. Antonie Van Leeuwenhoek 106:331–345CrossRefPubMedGoogle Scholar
  49. Paul VJ, Ritson-Williams R, Sharp K (2011) Marine chemical ecology in benthic environments. Nat Prod Rev 28:345–387CrossRefGoogle Scholar
  50. Paz Z, Komon-Zelazowska M, Druzhinina IS, Aveskamp MM, Shnaiderman A, Aluma Y, Carmeli S, Ilan M, Yarden O (2010) Diversity and potential antifungal properties of fungi associated with a Mediterranean sponge. Fungal Divers 42:17–26CrossRefGoogle Scholar
  51. Pérez-Portela R, Turon X (2007) Prey preferences of the polyclad flatworm Prostheceraeus roseus among Mediterranean species of the ascidian genus Pycnoclavella. Hydrobiologia 592:535–539CrossRefGoogle Scholar
  52. Pérez-Portela R, Duran S, Palacin C, Turon X (2007) The genus Pycnoclavella (Ascidiacea) in the Atlanto- Mediterranean region: a combined molecular and morphological approach. Invertebr Syst 21:187–205CrossRefGoogle Scholar
  53. Pisut DP, Pawlik JR (2002) Anti-predatory chemical defenses of ascidians: secondary metabolites or inorganic acids? J Exp Mar Biol Ecol 270:203–214CrossRefGoogle Scholar
  54. Rowan R (1998) Diversity and ecology of zooxanthellae on coral reefs. J Phycol 34:407–417CrossRefGoogle Scholar
  55. Schmitt S, Tsai P, Bell J, Fromont J, Ilan M, Lindquist N, Perez T, Rodrigo A, Schupp PJ, Vacelet J, Webster NS, Hentschel U, Taylor MW (2012) Assessing the complex sponge microbiota: core, variable and species-specific bacterial communities in marine sponges. ISME J 6:564–576PubMedCentralCrossRefPubMedGoogle Scholar
  56. Shenkar N, Swalla BJ (2011) Global diversity of Ascidiacea. PLoS ONE 6:e20657PubMedCentralCrossRefPubMedGoogle Scholar
  57. Sun H-H, Mao W-J, Jiao J-Y, Xu J-C, Li H-Y, Chen Y, Qi X-H, Chen Y-L, Xu J, Zhao C-Q, Hou Y-J, Yang Y-P (2011) Structural characterization of extracellular polysaccharides produced by the marine fungus Epicoccum nigrum JJY-40 and their antioxidant activities. Mar Biotechnol 13:1048–1055CrossRefPubMedGoogle Scholar
  58. Suryanarayanan TS (2012) The diversity and importance of fungi associated with marine sponges. Bot Mar 55:553–564CrossRefGoogle Scholar
  59. Tait E, Carman M, Sievert SM (2007) Phylogenetic divrsity of bacteria associated with ascidians in Eel Pond (Woods Hole, Massachusetts, USA). J Exp Mar Biol Ecol 342:138–146CrossRefGoogle Scholar
  60. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729PubMedCentralCrossRefPubMedGoogle Scholar
  61. Tarjuelo I, López-Legentil S, Codina M, Turon X (2002) Defence mechanisms of adults and larvae of colonial ascidians: patterns of palatability and toxicity. Mar Ecol Prog Ser 235:103–115CrossRefGoogle Scholar
  62. Taylor MW, Schupp P, Dahllof I, Kjelleberg S, Steinberg PD (2004) Host specificity in marine sponge-associated bacteria, and potential implications for marine microbial diversity. Environ Microbiol 6:121–130CrossRefPubMedGoogle Scholar
  63. Thacker RW, Freeman CJ (2012) Sponge-microbe symbioses: recent advances and new directions. Adv Mar Biol 62:57–111CrossRefPubMedGoogle Scholar
  64. Tianero MDB, Kwan JC, Wyche TP, Presson AP, Koch M, Barrows LR, Bugni TS, Schmidt EW (2015) Species specificity of symbiosis and secondary metabolism in ascidians. ISME J 9:615–628CrossRefPubMedGoogle Scholar
  65. Van Bressem M-F, Raga JA, Di Guardo G, Jepson PD, Duignan PJ, Siebert U, Barrett T, de Oliveira Santos MC, Moreno IB, Siciliano S, Aguilar A, Van Waerebeek K (2009) Emerging infectious diseases in cetaceans worldwide and the possible role of environmental stressors. Dis Aquat Org 86:143–157CrossRefPubMedGoogle Scholar
  66. Webster NS (2007) Sponge disease: a global threat? Environ Microbiol 9:1363–1375CrossRefPubMedGoogle Scholar
  67. Webster NS, Taylor MW (2012) Marine sponges and their microbial symbionts: love and other relationships. Environ Microbiol 14:335–346CrossRefPubMedGoogle Scholar
  68. Wheat LJ, Bartlett M, Ciccarelli M, Smith JW (1984) Opportunistic Scopulariopsis pneumonia in an immunocompromised host. South Med J 77:1608–1609CrossRefPubMedGoogle Scholar
  69. White TJ, Bruns T, Lee S, Taylor J (1990) Aplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications. Academic, New YorkGoogle Scholar
  70. Wiese J, Ohlendorf B, Blümel M, Schmaljohann R, Imhoff J (2011) Phylogenetic identification of fungi isolated from the marine sponge Tethya aurantium and identification of their secondary metabolites. Marine Drugs 9:561–585PubMedCentralCrossRefPubMedGoogle Scholar
  71. Williamson B, Tudzynski B, Tudzynski P, Van Kan JA (2007) Botrytis cinerea: the cause of grey mould disease. Mol Plant Pathol 8:561–580CrossRefPubMedGoogle Scholar
  72. Xin ZH, Tian L, Zhu TJ, Wang WL, Du L, Fang YC, Gu QQ, Zhu WM (2007) Isocoumarin derivatives from the sea squirt-derived fungus Penicillium stoloniferum QY2-10 and the halotolerant fungus Penicillium notatum B-52. Arch Pharm Res 30:816–819CrossRefPubMedGoogle Scholar
  73. Yarden O (2014) Fungal association with sessile marine invertebrates. Front Microbiol 5:228PubMedCentralCrossRefPubMedGoogle Scholar
  74. Yarden O, Ainsworth TD, Roff G, Leggat W, Fine M, Hoegh-Guldberg O (2007) Increased prevalence of ubiquitous ascomycetes in an acropoid coral (Acropora formosa) exhibiting symptoms of brown band syndrome and skeletal eroding band. Appl Environ Microbiol 73:2755–2757PubMedCentralCrossRefPubMedGoogle Scholar
  75. Yu ZG, Lang G, Kajahn I, Schmaljohann R, Imhoff JF (2008) Scopularides A and B, cyclodepsipeptides from a marine sponge-derived fungus, Scopulariopsis brevicaulis. J Nat Prod 71:1052–1054CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Susanna López-Legentil
    • 1
    Email author
  • Patrick M. Erwin
    • 1
  • Marta Turon
    • 2
  • Oded Yarden
    • 3
  1. 1.Department of Biology and Marine Biology, and Center for Marine ScienceUniversity of North Carolina WilmingtonWilmingtonUSA
  2. 2.Center for Advanced Studies of Blanes (CEAB-CSIC)BlanesSpain
  3. 3.Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and EnvironmentThe Hebrew University of JerusalemRehovotIsrael

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