Mycol Progress (2016) 15: 18 DOI 10.1007/s11557-016-1161-4 ORIGINAL ARTICLE A new species of Exophiala associated with roots Jose G. Maciá-Vicente1,2 & Kyriaki Glynou1,2 & Meike Piepenbring1,2 Received: 9 October 2015 /Revised: 14 December 2015 /Accepted: 20 January 2016 /Published online: 1 February 2016 # German Mycological Society and Springer-Verlag Berlin Heidelberg 2016 Abstract A new species of the genus Exophiala Introduction (Herpotrichiellaceae, Ascomycota), Exophiala radicis,isde- scribed. The description is based on five strains isolated as The genus Exophiala constitutes a polymorphic group of asco- endophytes from roots of the brassicaceous plant mycetous fungi within the family Herpotrichiellaceae Microthlaspi perfoliatum s.l., collected at different localities (Chaetothyriales). It includes dematiaceous anamorphic species in Europe. As evidenced by phylogenetic analyses of regions characterized by annellidic conidiogenesis and frequent yeast- of the ribosomal DNA [the small and large subunits, and the like states. Its known teleomorphs belong in the genus internal transcribed spacers (ITS)] and the translation elonga- Capronia (Carmichael 1967;Hironagaetal.1981; De Hoog tion factor 1-α,theβ-tubulin, and the actin genes, the new et al. 2011). Most studies on Exophiala species focus on their species is closely related to Exophiala tremulae and Exophiala importance as etiologic agents of disease in animals and humans equina. E. radicis differs from E. tremulae morphologically (Richards et al. 1978; Zeng and de Hoog 2008; De Hoog et al. by the shape and size of their conidia. A comparison of ITS 2011; Najafzadeh et al. 2013; Wen et al. 2015), and they include sequences of E. radicis with GenBank records suggests that assessments of their occurrence in anthropogenic habitats, such the species has a wide distribution in the northern hemisphere, as bathroom facilities or bottled water, which constitute poten- and that it is commonly associated with living plant roots, tial sources of infection (Iwatsu et al. 1991; Matos et al. 2002; indicating potential adaptations to this substrate. Ávila et al. 2005;Isolaetal.2013). However, the pathogenic lifestyle of Exophiala species is opportunistic, and members of the genus are frequently isolated from natural environments Keywords Chaetothyriales . Endophytes . Exophillic acid . independent of potential animal hosts, such as bulk soil, biolog- Roots . salmonis-clade ical crusts, rock surfaces, air, natural water masses, the rhizo- sphere, and plant tissues (Addy et al. 2005; Julou et al. 2005; Bates et al. 2006; Neubert et al. 2006; Bukovská et al. 2010;De Section Editor: Roland Kirschner Hoog et al. 2011; Ferrari et al. 2011). Such diversity of ecolog- Electronic supplementary material The online version of this article ical sources indicates that the species of Exophiala have versa- (doi:10.1007/s11557-016-1161-4) contains supplementary material, tile lifestyles with adaptations to thrive in multiple habitats. This which is available to authorized users. is reflected in a pleomorphism in the genus, with species displaying synanamorphs (e.g., Phaeococcomyces or * Jose G. Maciá-Vicente [email protected] Phialophora;Untereineretal.1995; De Hoog et al. 2011)that include the production of budding cells (yeasts) by many spe- cies (Zeng and de Hoog 2008). The polymorphic nature of Exophiala species make them difficult to be identified by their 1 Institute of Ecology, Evolution and Diversity, Goethe University Frankfurt, Max-von-Laue-Str. 13, D-60438 Frankfurt am morphology alone and, therefore, sequencing of nuclear regions Main, Germany of the ribosomal DNA is considered as necessary for identifica- 2 Integrative Fungal Research Cluster (IPF), Georg-Voigt-Str. 14-16, tion of species (Untereiner and Naveau 1999; Bates et al. 2006; 60325 Frankfurt am Main, Germany Zeng and de Hoog 2008; De Hoog et al. 2011). 18 Page 2 of 12 Mycol Progress (2016) 15: 18 In a recent study, the secondary metabolites produced in lactophenol blue, using a Zeiss Axio Lab.A1 microscope and culture by five isolates of an Exophiala species were analyzed an Axiocam ERc 5 s camera (Zeiss, Hamburg, Germany). (Cheikh-Ali et al. 2015). The strains were isolated as endo- Microscopic structures were drawn from preparations phytes from roots of Microthlaspi perfoliatum s.l. (L.) F.K. mounted with distilled water and observed in a Zeiss Meyer (Brassicaceae; Ali et al. 2016) growing at different Axioscop 2 plus, aided by a scale in the ocular and a scaled localities in Europe, and all produced a set of natural products grid. Radial growth rates of colonies were measured periodi- similar to exophillic acid, a metabolite previously described cally from cultures inoculated using mycelium on a 5-mm- from Exophiala pisciphila McGinnis & Ajello (McGinnis and diameter agar plug and incubated at either 25 °C or 37 °C. Ajello 1974; Ondeyka et al. 2003). Differences in their pro- files of secondary metabolite production could be linked to DNA amplification and sequencing morphological differences on their cultures: strains with flat and slimy colonies produced chemical derivatives containing Sequences of the ITS regions 1 and 2 and the 5.8S rDNA of a monosaccharide moiety (β-D-glucopyranosyl), while strains the strains in this study are available in GenBank (Table 1; with dome-shaped colonies and aerial mycelium had similar Glynou et al. 2016). We amplified and sequenced five addi- derivatives but without the monosaccharide (Cheikh-Ali et al. tional nuclear loci in polymerase chain reactions containing 2015). In spite of their morphological and chemical differ- 1 μL of DNA template, 2-mM MgCl2,0.2-mMdNTPs, ences, all strains are considered to pertain to the same species 0.3 μM of each primer, and 0.5 U Taq polymerase (VWR according to similarities in their micromorphology and inter- International, Darmstadt, Germany). A part of the large sub- nal transcribed spacer region (ITS) sequences. However, they unit (LSU) of the rDNA was amplified using the primer pair could not be classified in any known species of Exophiala LR0R/LR7 (Hopple and Vilgalys 1994) with the following according to their morphological traits and phylogenetic affin- temperature cycles: 94 °C for 4 min, 35 cycles of 94 °C for ities. Here, we provide a formal description of these strains 1 min, 48 °C for 45 s, and 72 °C for 2 min, and a final step of based on morphological, molecular, and ecological data. 72 °C for 5 min. The partial small subunit (SSU) of the rDNA was amplified with primers NSSU131/NS24 (Kauff and Lutzoni 2002) using the above cycling conditions, but with Materials and methods an annealing temperature of 52 °C. The partial translation elongation factor 1-α (TEF1-α)andβ-tubulin (β-tub)genes Strains and culture conditions were amplified with primer pairs Ef1-728F/Ef1-986R (Carbone and Kohn 1999) and Bt2a/Bt2b (Glass and The strains in this study were isolated in 2013 as root endo- Donaldson 1995), respectively, as in De Hoog et al. (2011). phytes from several specimens of Microthalspi perfoliatum The partial actin gene (act1) was amplified with primers s.l. collected at different localities in Europe (Glynou et al. LPW17499/LPW17500 (Woo et al. 2013) with the following 2016). Strain P1095 was isolated from a plant in eastern temperature cycles: 94 °C for 4 min, 35 cycles of 94 °C for France, strains P1860 and P1910 were isolated from individ- 30 s, 55 °C for 30 s, and 72 °C for 45 s, and one step of 72 °C ual plants within the same population in Bulgaria, and strains for 5 min. Amplicons were purified with the EZNA Cycle P2772 and P2854 originated from different plant populations Pure Kit (Omega Bio-Tek, Norcross, GA, USA) and se- in Germany. The reference ex-type strain of Exophiala quenced at GATC Biotech (Konstanz, Germany). Sequences tremulae W. Wang (CBS 129355; Crous et al. 2011)was of the SSU, TEF1-α, β-tub,andact1 were likewise obtained obtained from the KNAW-CBS Fungal Biodiversity Centre for the reference strain E. tremulae CBS 129355. All se- (CBS). Strains are maintained on corn meal agar slants cov- quences generated in this work have been deposited in ered with mineral oil at room temperature at the IPF GenBank (see Table 1 for accession numbers). (Integrative Fungal Research) collection hosted at the Goethe University (Frankfurt am Main, Germany). Phylogenetic analyses Duplicate cultures are also deposited in the CBS culture col- lection (Utrecht, The Netherlands). Phylogenetic analyses were performed separately for every The strains were grown on 2 % malt extract agar (MEA, locus using maximum likelihood (ML) and Bayesian phylo- Applichem, Darmstadt, Germany) and potato dextrose agar genetic inference. A selection of available representative se- (PDA, Applichem). They were cultured in triplicate for mor- quences from strains of the closest Exophiala species within phological examinations and growth measurements. For the salmonis clade of Exophiala (De Hoog et al. 2011)was microscopic observations, samples were prepared as included as a reference (Table 1). Sequences were aligned described in De Hoog et al. (2011) and observed after 7 to using the G-INS-i algorithm of MAFFT v7.123b (Katoh and 14 days, or directly mounted in squash preparations from Standley 2013) and then trimmed with Gblocks v0.91b older cultures. Micrographs were taken on slides stained with (Castresana 2000). For ML phylogenies, we used RAxML Mycol Progress (2016) 15: 18 Table 1 Reference strains of Exophiala included in the phylogenetic analyses GenBank accessions Proposed Original name Strainb Source Location ITS LSU SSU TEF1-αβ-tub act1 Reference classificationa Exophiala aquamarina Exophiala CBS 119918 (T) Skin of leafy sea dragon USA NR_111626 - JN856012 - JN112434 JN112388 De Hoog et al. aquamarina (2011) Exophiala brunnea Exophiala brunnea CBS 587.66 (T) Litter of Acacia karoo South Africa NR_119959 - JN856013 JN128783 JN112442 JN112393 De Hoog et al. (2011) Exophiala cancerae Exophiala cancerae CBS 120420 (T) Diseased mangrove Brazil HQ659023 - - JN128800 JN112444 JN112394 De Hoog et al.