, Volume 174, Issue 5–6, pp 397–408 | Cite as

Cladosporium cladosporioides LPSC 1088 Produces the 1,8-Dihydroxynaphthalene-Melanin-Like Compound and Carries a Putative pks Gene

  • Carla Llorente
  • Alejandra Bárcena
  • José Vera Bahima
  • Mario C. N. Saparrat
  • Angélica M. Arambarri
  • M. Fernanda Rozas
  • María V. Mirífico
  • Pedro A. Balatti


Cladosporium cladosporioides is a dematiaceous fungus with coloured mycelia and conidia due to the presence of dark pigments. The purpose of this study was to characterize the dark pigments synthetized by Cladosporium sp. LPSC no. 1088 and also to identify the putative polyketide synthase (pks) gene that might be involved in the pigment biosynthesis. Morphological as well as molecular features like the ITS sequence confirmed that LPSC 1088 is Cladosporium cladosporioides. UV-visible, Fourier Transform Infrared (FTIR) and Electron Spin Resonance (ESR) spectroscopy analysis as well as melanin inhibitors suggest that the main dark pigment of the isolate was 1,8 dihydroxynaphthalene (DHN)-melanin-type compound. Two commercial fungicides, Difenoconazole and Chlorothalonil, inhibited fungal growth as well as increased pigmentation of the colonies suggesting that melanin might protect the fungus against chemical stress. The pigment is most probably synthetized by means of a pentaketide pathway since the sequence of a 651 bp fragment, coding for a putative polyketide synthase, is highly homologous to pks sequences from other fungi.


DHN melanin Cladosporium cladosporioides Fungicides Polyketide synthase pks 



We thank Bucsinszky, A. M. M., Rollán, C. and Ronco, L. for field and laboratory assistance and Gennaro, A. M., Ph D, for performing the ESR determination. The ESR analysis was carried out at INTEC (CONICET-Universidad Nacional del Litoral), Santa Fe, Argentina. Bárcena, A. is recipient of a scholarship from the Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CICPBA), Argentina. Vera Bahima, J. is recipient of a scholarship from the CONICET, Argentina. Arambarri, A. M., Mirífico, M. V. and Saparrat, M. C. N. are researchers from the CONICET, Argentina. Rozas, M. F. is a CONICET technician. Balatti, P. A. is a researcher from the CICPBA, Argentina. This research was partially supported by grants from CONICET (PIP 1422), CICPBA and UNLP, Argentina.


  1. 1.
    Braun U, Crous PW, Dugan FM, Groenewald JZ, Hoog GS. Phylogeny and taxonomy of Cladosporium-like hyphomycetes, including Davidiella gen. nov., the teleomorph of Cladosporium s.str. Mycol Progress. 2003;2:3–18.CrossRefGoogle Scholar
  2. 2.
    Dugan FM, Schubert K, Braun U. Check-list of Cladosporium names. Schlechtendalia. 2004;11:1–103.Google Scholar
  3. 3.
    Bensch K, Groenewald JZ, Dijksterhuis J, Starink-Willemse M, Andersen B, Summerell BA, Shin HD, Dugan FM, Schroers HJ, Braun U, Crous PW. Species and ecological diversity within the Cladosporium cladosporioides complex (Davidiellaceae, Capnodiales). Stud Mycol. 2010;67:1–94.PubMedCrossRefGoogle Scholar
  4. 4.
    Crous PW, Braun U, Schubert K, Groenewald JZ. Delimiting Cladosporium from morphologically similar genera. Stud Mycol. 2007;58:33–56.PubMedCrossRefGoogle Scholar
  5. 5.
    Ellis MB. Dematiaceus hyphomycetes. Kew England: Commonwealth Mycological Institute; 1971.Google Scholar
  6. 6.
    Poutahidis T, Angelopoulou K, Karamanavi E, Polizopouloux ZS, Doulberis M, Latsari M, Kaldrymidou E. Mycotic encephalitis and nephritis in a dog due to infection with Cladosporium cladosporioides. J Comp Pathol. 2009;140:59–63.PubMedCrossRefGoogle Scholar
  7. 7.
    Pilawa B, Buszman E, Gondzik AS, Wilczynski S, Zdybel M, Witoszynska T, Wilczok T. Effect of pH on paramagnetic centers in Cladosporium cladosporioides melanin. Acta Phys Pol, A. 2005;108:147–50.Google Scholar
  8. 8.
    Bell AA, Wheeler MH. Biosynthesis and functions of fungal melanins. Annu Rev Phytopathol. 1986;24:411–51.CrossRefGoogle Scholar
  9. 9.
    Henson JM, Butler MJ, Day AW. The dark side of the mycelium: melanins of phytopathogenic fungi. Annu Rev Phytopathol. 1999;37:447–71.PubMedCrossRefGoogle Scholar
  10. 10.
    Butler MJ, Gardiner RB, Day AW. Melanin synthesis by Sclerotinia sclerotiorum. Mycologia. 2009;101:296–304.PubMedCrossRefGoogle Scholar
  11. 11.
    Saparrat MCN, Fermoselle G, Stenglein S, Aulicino M, Balatti P. Pseudocercospora griseola causing angular leaf spot on Phaseolus vulgaris produces 1,8-dihydroxynaphthalene-melanin. Mycopathologia. 2009;1:41–7.CrossRefGoogle Scholar
  12. 12.
    Latgé JP, Bouziane H, Diaquin M. Ultrastructure and composition of the conidial wall of Cladosporium cladosporioides. Can J Microbiol. 1988;34:1325–9.PubMedCrossRefGoogle Scholar
  13. 13.
    Chodurek E, Latocha M, Kurkiewicz S, Buszman E, Swiatkowska L, Wilczok T. Chemical characteristics of melanin from Cladosporium cladosporioides. Bull Pol Acad Sci Biol Sci. 1998;46:51–8.Google Scholar
  14. 14.
    Tu YG, Xie MY, Sun YZ, Tian YG. Structural characterization of melanin from black-bone silky fowl (Gallus gallus domesticus Brisson). Pigment Cell Melanoma Res. 2008;22:134–6.PubMedCrossRefGoogle Scholar
  15. 15.
    Buszman E, Wiewióra A, Andrzejczyk J, Wilczok T. Physicochemical properties of melanin isolated from Cladosporium cladosporioides. Curr Top Biophys. 1992;16:73–6.Google Scholar
  16. 16.
    Dadachova E, Bryan RA, Huang X, Moadel T, Schweitzer AD, Aisen P, Nosanchuk JD, Casadevall A. Ionizing radiation changes the electronic properties of melanin and enhances the growth of melanized fungi. PLoS ONE. 2007;5:1–13.Google Scholar
  17. 17.
    Gadd GM. Effects of media composition and light on colony differentiation and melanin synthesis in Microdochium bolleyi. Trans Br Mycol Soc. 1982;78:115–22.CrossRefGoogle Scholar
  18. 18.
    Suryanarayanan TS, Ravishankar JP, Venkatesan G, Murali TS. Characterization of the melanin pigment of a cosmopolitan fungal endophyte. Mycol Res. 2004;108:974–8.PubMedCrossRefGoogle Scholar
  19. 19.
    Bashyal BM, Chand R, Kushwaha C, Sen D, Prasad LC, Joshi AK. Association of melanin content with conidiogenesis in Bipolaris sorokiniana of barley (Hordeum vulgare L.). World J Microbiol Biotechnol. 2010;26:309–16.CrossRefGoogle Scholar
  20. 20.
    Selvakumar P, Rajasekar S, Periasamy K. Isolation and characterization of melanin pigment from Pleurotus cystidiosus (telomorph of Antomycopsis macrocarpa). World J Microbiol Biotechnol. 2008;24:2125–31.CrossRefGoogle Scholar
  21. 21.
    Saparrat MCN, Estevez JM, Troncozo MI, Arambarri A, Balatti P. In-vitro depolymerization of Scutia buxifolia leaf-litter by a dominant Ascomycota Ciliochorella sp. Int Biodeterior Biodegrad. 2010;64:262–6.CrossRefGoogle Scholar
  22. 22.
    Babitskaya VG, Shcherba VV, Filimonova TV, Grigorchuk EA. Melanin pigments from the fungi Paecilomyces variotii and Aspergillus carbonarius. Appl Biochem Microbiol. 2000;36:128–33.CrossRefGoogle Scholar
  23. 23.
    Tavzes C, Silc F, Kladnik A, Fackler K, Messner K. Enzymatic degradation of mould stains on paper analysed by colorimetry and DRIFT-IR spectroscopy. Int Biodeterior Biodegrad. 2009;63:873–9.CrossRefGoogle Scholar
  24. 24.
    Griffith GW, Easton GL, Detheridge A, Roderick K, Edwards A, Worgan HJ, Nicholson J, Perkins WT. Copper deficiency in potato dextrose agar causes reduced pigmentation in cultures of various fungi. FEMS Microbiol Lett. 2007;276:165–71.PubMedCrossRefGoogle Scholar
  25. 25.
    Koné D, Badou OJ, Bomisso EL, Camara B, Ake S. Activités in-vitro de différents fongicides sur la croissance chez Mycosphaerella fijiensis var. difformis Stover et Dickson, Cladosporium musae Morelet et Deightoniella torulosa (Syd.) Ellis, parasites isolés de la phyllosphère des bananiers en Côte-d’Ivoire. Biologies. 2009;332:448–55.PubMedCrossRefGoogle Scholar
  26. 26.
    Bornet B, Branchard M. Nonanchored inter simple sequence repeat (ISSR) markers: reproducible and specifics tools for genome fingerprinting. Plant Mol Biol Rep. 2001;19:209–15.CrossRefGoogle Scholar
  27. 27.
    White TJ, Bruns T, Lee S, Taylor JW. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols: a guide to methods and applications. San Diego: Academic Press; 1990. p. 315–22.Google Scholar
  28. 28.
    Bingle TJS, Lewis EH, Colin ML. Ketosynthase domain probes identify two subclasses of fungal polyketide synthase genes. Fungal Genet Biol. 1999;26:209–23.PubMedCrossRefGoogle Scholar
  29. 29.
    Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Nat Acad Sci USA. 1977;74:5463–7.PubMedCrossRefGoogle Scholar
  30. 30.
    Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucl Acids Res. 1994;22:4673–80.PubMedCrossRefGoogle Scholar
  31. 31.
    Varga J, Rigó K, Kocsubé S, Farkas B. Diversity of polyketide synthase gene sequences in Aspergillus species. Res Microbiol. 2003;154:593–600.PubMedCrossRefGoogle Scholar
  32. 32.
    Matuszczyk M, Buszman E, Pilawa B, Witoszynska T, Wilczok T. Cd2+ effect on free radicals in Cladosporium cladosporioides-melanin tested by EPR spectroscopy. Chem Phys Lett. 2004;394:366–71.CrossRefGoogle Scholar
  33. 33.
    Buszman E, Pilawa B, Zdybel M, Wilczynski S, Gondzik A, Witoszynska T, Wilczok T. EPR examination of Zn2+ and Cu2+ binding by pigmented soil fungi Cladosporium cladosporioides. Sci Total Environ. 2006;363:195–205.PubMedCrossRefGoogle Scholar
  34. 34.
    Katritzky AR, Akhmedov NG, Denisenko SN, Denisko OV. 1H NMR spectroscopic characterization of solutions of Sepia melanin, Sepia melanin free acid and human hair melanin. Pigment Cell Res. 2002;15:93–7.PubMedCrossRefGoogle Scholar
  35. 35.
    Pihet M, Vandeputte P, Tronchin G, Renier G, Saulnier P, Georgeault S, Mallet R, Chabasse D, Symoens F, Bouchara JP. Melanin is an essential component for the integrity of the cell wall of Aspergillus fumigatus conidia. BMC Microbiol. 2009;9:177–87.PubMedCrossRefGoogle Scholar
  36. 36.
    Crowe JD, Olsson S. Induction of laccase activity in Rhizoctonia solani by antagonistic Pseudomonas fluorescens strains and a range of chemical treatments. Appl Environ Microbiol. 2001;67:2088–94.PubMedCrossRefGoogle Scholar
  37. 37.
    Icenhour CR, Kottom TJ, Limper AH. Evidence for a melanin cell wall component in Pneumocystis carinii. Infect Immun. 2003;71:5360–3.PubMedCrossRefGoogle Scholar
  38. 38.
    Seto Y, Kogami Y, Shimanuki T, Takahashi K, Matsuura H, Yoshihara T. Production of phleicrome by Cladosporium phlei as stimulated by diketopiperadines of Epichloe typhina. Biosci Biotechnol Biochem. 2005;69:1515–9.PubMedCrossRefGoogle Scholar
  39. 39.
    Singaravelan N, Grishkan I, Beharav A, Wakamatsu K, Ito S, Nevo E. Adaptive melanin response of the soil fungus Aspergillus niger to UV radiation stress at ‘‘Evolution Canyon’’, Mount Carmel, Israel. PLoS ONE. 2008;3(8):e2993. doi: 10.1371/journal.pone.0002993.PubMedCrossRefGoogle Scholar
  40. 40.
    Thywißen A, Heinekamp T, Dahse HM, Schmaler Ripcke J, Nietzsche S, Zipfel PF, Brakhage AA. Conidial dihydroxynaphthalene melanin of the human pathogenic fungus Aspergillus fumigatus interferes with the host endocytosis pathway. Front Microbiol. 2011;2:1–12. doi: 10.3389/fmicb.2011.00096.Google Scholar
  41. 41.
    Sauer M, Lu P, Sangari R, Kennedy S, Polishook J, Bills G, An Z. Estimating polyketide metabolic potencial among non-sporulating fungal endophytes of Vaccinium macrocarpon. Mycol Res. 2002;106:460–70.CrossRefGoogle Scholar
  42. 42.
    Nicholson TP, Rudd BAM, Dawson M, Lazarus CM, Simpson TJ, Cox RJ. Design and utility of oligonucleotide gene probes for fungal polyketide synthases. Chem Biol. 2001;8:157–78.PubMedCrossRefGoogle Scholar
  43. 43.
    Kroken S, Glass NL, Taylor JW, Yoder OC, Turgeon BG. Phylogenomic analysis of type I polyketide synthase genes in pathogenic and saprobic ascomycetes. Proc Nat Acad Sci USA. 2003;100:15670–5.PubMedCrossRefGoogle Scholar
  44. 44.
    Schmitt I, Martín MP, Kautz S, Lumbsch HT. Diversity of non-reducing polyketide synthase genes in the Pertusariales (lichenized Ascomycota): a phylogenetic perspectiva. Phytochemistry. 2005;66:1241–53.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Carla Llorente
    • 1
  • Alejandra Bárcena
    • 1
  • José Vera Bahima
    • 2
  • Mario C. N. Saparrat
    • 3
    • 4
    • 5
  • Angélica M. Arambarri
    • 4
  • M. Fernanda Rozas
    • 6
  • María V. Mirífico
    • 6
    • 7
  • Pedro A. Balatti
    • 3
    • 5
    • 8
  1. 1.Instituto de Fisiología Vegetal (INFIVE), CCT-La Plata-Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)Universidad Nacional de La Plata (UNLP)La PlataArgentina
  2. 2.Facultad de Ciencias Agrarias y ForestalesCentro de Investigaciones de Fitopatología (CIDEFI), UNLPLa PlataArgentina
  3. 3.INFIVE, UNLP-CCT-La Plata-CONICETLa PlataArgentina
  4. 4.Facultad de Ciencias Naturales y MuseoInstituto de Botánica Spegazzini, UNLPLa PlataArgentina
  5. 5.Facultad de Ciencias Agrarias y ForestalesCátedra de Microbiología Agrícola, UNLPLa PlataArgentina
  6. 6.Facultad de Ciencias Exactas, UNLP, CCT-La Plata-CONICETInstituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA)La PlataArgentina
  7. 7.Facultad de Ingeniería, UNLPÁrea Departamental Ingeniería QuímicaLa PlataArgentina
  8. 8.Facultad de Ciencias Agrarias y Forestales, UNLPCIDEFILa PlataArgentina

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