ACtA MYCoLoGICA Vol. 43 (1): 67–70 2008 Cephalotrichum stemonitis as a biofilm inhabitant in the gold mine in Poland ANDRZEJ CHLEBICKI Department of Mycology, W. Szafer Institute of Botany, Polish Academy of Sciences Lubicz 46, PL-31-512 Kraków, ibchlebick@ib-krakow.pl Chlebicki A.: Cephalotrichum stemonitis as a biofilm inhabitant in gold mine in Poland. Acta Mycol. 43 (1): 67–70, 2008. Cephalotrichum stemonitis and its synanamorph Echinobotryum atrum isolated from bacterial biofilm is presented. Key words: mine fungi, Cephalotrichum, biofilm, distribution INtRoDuCtIoN Dry-spored synnematous anamorphs from the form-genus Cephalotrichum Link are the asexual states of Microascaceae. However so far a teleomorph state is known for no species of this genus (Abbott 2000). Microascaceae include five genera: Microascus, Kernia, Petriella, Pseudoalescheria and Lophotrichus (Abbott et al. 2002) as well as eight anamorphic genera containing about 50 species (Abbott 2000). Abbott (2000) analyzed the subunit 18S rDNA for 34 taxa of Microascaceae. He obtained Microascus clade consisting of Microascus longirostris, M. nidicola, M. cirrhosus, M. trigonosporus, Cephalotrichum stemonitis, Wardomyces anomalus, Cephalotrichum cylindricum and Microascus brevicaulis. It indicate that the genus Microascus can be recognized as hypothetical teleomorph state for Cephalotrichum stemonitis. In GenBank there are available 3 sequences of ribosomal RNA genes (5.8S rRNA, 28S rRNA and large subunit rRNA) of Doratomyces stemonitis (in fact Cephalotrichum stemonitis) with access numbers: AF400852, EF029213 and AJ608983. Synnemata of Cephalotrichum producing chains of powdery conidia are relatively large with ‘bootle brush’ or “feather” appearance. ovoid conidia are produced from annelidic conidiogenous cells covering sporogonous area. Cephalotrichum synnemata are adapted for dispersion not only by air currents but also by insects (Abbott 2000, 2002). Habitat of Cephalotrichum fungi includes soil, compost, wood, wood treated with fungicide, herbaceous stems, oat seeds, decaying plant material and dung, finger nail, sawdust and straw used for growing shiitake, airborne contaminant of wheat-straw agar plate, egg of gypsy moth (Lymantaria dispar), roots of potato, 68 A. Chlebicki basidiomycete detritus, manore pile, bronchial washing, right feel, leaves of needleleafed tree (Abbott 2000). C. stemonitis was noted in coyote and rat dung, indoor air of honeybee (Apis mellifera) overwintering facility, cone of white spruce, sandy soil, decayed wood of white spruce, soil of elm woods, agricultural soil. It is also important colonizer of pPVC buried in soil (Sabev et al. 2006). these authors mentioned it as Doratomyces spp. in the article, but in GenBank it is more precisely determined as D. stemonitis. Malloch and Hubart (1987) described unnamed species of Microascus from Ramoil Cave. It means that fungi from this group can inhabit also underground environments. Fassatiova (1970) noted C. stemonitis on wood in uranium mine in Czech Republic. StuDY AREA AND MEtHoDS Gray biofilms of rock-inhabiting bacteria in Gertruda Adit in a closed gold mine located in Złoty Stok in Lower Silesia were chosen for microbial analysis. This mine posseses constant conditions such as low temperature ca 10º C, darkness, high humidity, high As concentration and other toxic substances (Chlebicki et al. 2005). So far 12 species of fungi were noted in this mine (Chlebicki et al. 2005; Chlebicki, Lorenc 2006). Bacteria with fungi were collected in sterile plastic tubes and refrigeratorated at 10º C. Fungal growth was performed on DRBC, RBC, YMA and PDA media. Inoculated media on Petri dishes were putted in incubator at 10º C. the morphological characters of the living fungi were examined in water and cotton blue in lactophenol using light microscopy (Nikon SMZ 1500, Nikon Labophot 2 and Nikon Eclipse 800). Microphotographs were taken with these microscopes equipped with a digital camera. For scanning electron microscope (SEM) studies mycelium was coated with gold, and photographed using a LEo 1430 VP Zeiss microscope with a working distance of ca 10 mm. Fungus-species nomenclature follows Abbott (2000). RESuLtS Cephalotrichum stemonitis (Pers.) Nees Magazin Ges. naturf. Freunde, Berlin 3: 20 (1809) Synonymy: Doratomyces stemonitis (Pers.) F.J. Morton & G. Sm., Mycol. Pap. 86: 70, 1963 Doratomyces stemonitis var. keratinolyticus (Dominik & Majchr.) Dominik & Majchr. Ekol. Pol., Ser. A 18: 603, 1970 Echinobotryum atrum Corda, Sturm’s Deutschl. Flora, III (Pilze) 3(12): 51 (1829) Periconia stemonitis Pers., Syn. meth. fung. (Göttingen): 687 (1801) Stysanus stemonitis (Pers.) Corda, Icon. fung. (Prague) 1: 22 (1837) Description: mycelium creamy-white to dark brown, after 7 days on PDA 17,5-21 mm diam., on RBC 17,0 mm diam., and on DRBC 13-17 mm, all in room temperature (Fig. 1A, B, D). First conidiophores of synanamorph – Echinonobotryum atrum – appeared throughout the mycelium after 12 days on PDA. Mycelium growing on RBC after two weeks produced rings of densely distributed synnemata of C. stemonitis in central part of the mycelium. Mycelium on PDA formed at the beginning E. Cephalotrichum stemonitis 69 atrum throughout the surface of dark brown colony and later numerous synnemata of C. stemonitis. Mycelium on YMA form brown and irregular colony patches (Fig. 1C). Synnemata 190-300 μm long, sterile part 130-210 μm, fertile part 60-120 μm long, (irregular heads) similar to feather (Fig. 2B, C) associated with synanamorph Echinobotryum atrum (Fig. 2A), conidiophores synnematous, brown, conidiogenous cells ampulliform, percurrent, conidia smooth (Fig. 2D), ovoid with pointed apex and truncate base 7-9,1 x 4,2-6,1 μm, pale brown. Material examined: on rock bacterial biofilm in Gertruda Adit in gold mine in Złoty Stok, Lower Silesia, Poland, 16 October 2006, coll. A. Chlebicki. First culture obtained on DRBC medium, then transferred to other media. DISCuSSIoN So far two species of the genus Cephalotrichum Link were noted in Poland. Dominik and Majchrowicz (1965) and Dominik (1970) described a new variety Doratomyces stemonitis var. keratinolyticus (Dominik & Majchr.) Dominik & Majchr. on the basis of specimen isolated from the soil. Conidia of this specimen are something smaller than typical variety, 4-6 x 2.5-4 μm and ‘very slightly rough’ (Dominik 1970). It resembles Cephalotrichum microsporum (Sacc.) P. M. Kirk. Moreover these authors did not mention the presence of Echinobotryum synanamorph which is diagnostic character of Cephalotrichum stemonitis. the next species Cephalotrichum putredinis (Corda) S. P. Abbott was reported by Dominik (1970) as Doratomyces albus (Szilvinyi) Dominik. unfortunately, these collections are not available for investigation. Presence of Cephalotrichum stemonitis in bacterial biofilm is accidental. However, as indicate informations of Fassatiova (1970) and Malloch and Hubart (1987), such fungi were noted in subterranean environments. Mille-Lindblom (2005), Hogan and Kolter (2002) and Kirkwood (2002) noted mostly antagonistic relation between fungi and bacteria. Fungi were always negatively affected by presence of bacteria. Penetration of fungal hyphae of Fusarium oxysporum was not observed where microcolonies of Pseudomonas were present, moreover Pseudomonas bacteria attached and colonized fungus hyphae (Bolwerk et al. 2003). Also growth of Cephalotrichum stemonitis was suppressed by bacteria from the genus Pseudomonas isolated from biofilm. REFERENCES Abbott S. P. 2000. Holomorph studies of the Microascaceae (PhD Dissertation). Edmonton, Alberta: univ. Alberta. 196 pp. Abbott S. P. 2002. Insects and other arthropods as agents of vector-dispersal in fungi. www.precisionenv. com./PDFS/AbbottInsectdispersal.pdf Abbott S. P., Lumley t. C., Sigler L. 2002. use of holomorph characters to delimit Microascus nidicola and M. soppii sp. nov., with notes on the genus Pithoascus. Mycologia 94 (2): 362–369. Bolwerk A. Lagopodi A. L., Wijfjes A. H. M., Lamers G. E. M., Chin-A-Woeng t. F. C., Lugtenberg B. J. J., Blomberg G. V. 2003. Interaction in the tomato rhizosphere of two Pseudomonas biocontrol strains with the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici. Mol. PlantMicrobe Interact. 16: 983–993. Chlebicki A., Godzik B., Lorenc M. W., Skłodowska A. 2005. Fungi and arsen-tolerant bacteria in the hypogean environment of an ancient gold mine in Lower Silesia SW Poland. Polish Bot. Stud. 19: 81–95. 70 A. Chlebicki Chlebicki A., Lorenc M. W. 2006. the troglophile fungus, Physisporinus vitreus on a mine wall at Złoty Stok, Poland. Polish. Bot. J. 22: 149–154. Dominik t. 1970. observations of new or noteworthy fungi from region of Szczecin. Zeszyty Naukowe Wyższej Szkoły Rolniczej w Szczecinie 32: 71–108. Dominik t., Majchrowicz I. 1965. Second contribution to the knowledge of keratynolytic and keratynophilic soil fungi in the region of Szczecin. Ekologia Polska 13: 415–447. Fassatiova o. 1970. Micromycetes inhabiting the mines of Přibram (Czechoslovakia). Česka Mykol. 24: 162–165. Hogan D. A., Kolter R. 2002. Pseudomonas-Candida interactions: and ecological role of virulence factors. Science 296: 2229–2232. Kirkwood M. L. 2002. Bacteria-fungi interactions: pathogenesis meets ecology. trends in Microbiology 10: 397–398. Malloch D., Hubart J. M. 1987. An undescribed species of Microascus from the Cave of Ramoiul. Canad. J. Bot. 65: 1281–1283. Mille-Lindblom C. 2005. Interaction between bacteria and fungi on aquatic detritus-causes and consequences. Acta universitatis upsaliensis. Digital comprehensive summaries of uppsala dissertations from the Faculty of Science and technology 46. 42 pp., uppsala. Sabev H. A, Handley P. S., Robson G. D. 2006. Fungal colonization of soil-buried plasticized polyvinyl chloride (pPVC) and the impact of incorporated biocides. Microbiology 152: 1731–1739. Cephalotrichum stemonitis zasiedlający bakteryjny biofilm w kopalni złota w Polsce Streszczenie Praca zawiera opis grzyba Cephalotrichum stemonitis i jego synanamorfy Echinobotryum atrum wyizolowanych z bakteryjnego biofilmu z Sztolni Gertrudy w Kopalni Złota w Złotym Stoku. Pierwsza informacja o tym gatunku podana z Polski przez t. Dominika i I. Majchrowicz jest niezbyt dokładna. Diagnostyczną cechą gatunku C. stemionitis jest obecność synanamorfy E. atrum o czym wymienieni autorzy nie wspominają. Podano również podstawowe dane o siedliskach i ekologii tego gatunku. Izolacja tego rzadkiego gatunku grzyba z bakteryjnego biofilmu nie była dotychczas notowana. Fig. 1. Colonies of Cephalotrichum stemonitis on different media: A – on RBC; B – on DRBC; C – on YMA; D – on PDA. Fig. 2. Morphology of Cephalotrichum stemonitis and its synanamorph – Echinobotryum atrum: A – conidia of E. atrum; scale bar = 10 µm; B – synnemata of C. stemonitis on DRBC medium; C – synnemata of C. stemonitis similar to feather; D – conidies of C. stemonitis.