ARTICLE 205
et al. et al. 2010). argillacea is an . R . 2013), R. eburnea et al richocomaceae Key words: environmental metagenomics Eurotiales Geosmithia indoor moulds Talaromyces T R. piperina ` . 2000). Thermotolerant and et al. 2000). (Houbraken IMA FUNGUS · VOLUME 4 · NO 2: 205–212 NO 2: 4 · · VOLUME FUNGUS IMA et al. 2010), and R. cylindrospora Thermostable enzymes offer several advantages for Thermostable enzymes offer species areAll previously known Rasamsonia aegroticola (Barton et al. 2010), R. argillacea ` . An increase in clinical reports suggests emerging pathogen, although its morphological similarity to Paecilomyces suggests it may have been misdiagnosed previously in clinical cases (Houbraken et al. 2010, De Ravin et al. 2011). biotechnology applications, including a reduction in the risk and of microbial contamination, prolonged storage viability, an increase in rates and yields enabled by high temperature et al. 2007). Turner processes (Haki & Rakshit 2003, (Houbraken species haveal. 2012, Houbraken et al. 2013). Rasamsonia and olive-brownroughened, paecilomyces-like conidiophores covering haveconidia, and ascomata with a scanty hyphal They have been detected in been observed in four species. as soil,Asia from substrates such America, Europe and North and canine human compost, conifer wood chips, indoor air, patients (Houbraken et al. 2012). optimum growththermotolerant or thermophilic, with *J / 6 PK / / #$P 4 / ! of their potential asthermophilic fungi are of concern because #$P 2009) andopportunistic pathogens (e.g. Robert & Casadevall of industrial interest for their ability to produce thermostable enzymes (Maheshwari et al. 2000). Several Rasamsonia species have been isolated from human patients, including R 2010), et et et al. eburnea . R Talaromyces Talaromyces Talaromyces and , composticola, (syn. . R , was recovered from one residence in Songkhla, Thailand, one residence in Songkhla, , was recovered from is a recently established genus including thermotolerant or is a recently established was not detected in the 454-pyrosequencing data for Thailand or data for was not detected in the 454-pyrosequencing byssochlamydoides (syn. argillacea (syn. Geosmithia . emersonii et al. (2010). . . R Geosmithia R R (Su & Cai 2012, Houbraken ) was established Trichocomaceae R. pulvericola Rasamsonia species. R. pulvericola
piperina . aegroticola, Eurotiales, R brevistipitata, ( . . . 2012). The genus currently consists of et al. 2012). ' #P ? ? 6 Rasamsonia In the course of a global survey of the indoor mycobiota, we sampled and analysed survey of the indoor mycobiota, we In the course of a global
Talaromyces eburneus), Talaromyces Rasamsonia cylindrospora (syn. Geosmithia cylindrospora), . Article info: " I $ ¤ NJ#*> ; I #K NJ#*> ~ I K | NJ#* and is morphologically characterised and compared phylogenetically with other members of the genus. characterised and compared phylogenetically and is morphologically characters such forms a clade with R. brevistipitata and shares morphological Rasamsonia pulvericola subglobose to ellipsoidal conidia. and never terverticillate conidiophores, and as usually biverticillate 6 The ITS sequence of was detected in Micronesia, probably representing another other countries, but a similar ITS sequence undescribed R. argillacea. A new species, and 454-pyrosequencing. and 454-pyrosequencing. © 2013 International Mycological Association © 2013 International Mycological conditions: distribute and transmit the work, under the following are free to share - to copy, You Attribution: Abstract: Joey B. Tanney, and Keith A. Seifert and Keith Tanney, Joey B. x 4 4 ; ; ?< / | / #; J/> ? I sp. nov., isolated from house dust from house isolated sp. nov., pulvericola Rasamsonia Non-commercial: No derivative works: Any of the above conditions can be waived if you get For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Nothing in this license impairs or restricts the author’s moral rights. permission from the copyright holder. . 2001) and share their work and living environments with al. 2001) and share their work and living The fungal biota may be a multitude of microorganisms. 6 inhabitants of biodeterioration, house plants as phyloplane (Flannigan or endophytes, or animals such as pets Humans spend up to 90 % of their time indoors (Klepeis Humans spend up to 90 % of their time INTRODUCTION 2011). In the north temperate societies of the western world In the north temperate societies 2011). methods, the that are most studied by conventional culturing 100 common fungi, indoor mycobiota is comprised of about species (Samson et and about 100 less frequently isolated in other climates al. 2010). Housing on other continents and investigations of the is less intensively sampled, and deeper 6 sequencing and alternative isolation techniques are only This paper reports on the isolation of a new fungal beginning. species in the recently recognised genus Rasamsonia, isolated from house dust samples that were also subjected to Amend 454-pyrosequencing by 6 6 argillacea), R byssochlamydoides), Talaromyces I#J KK$'L NJ#* JP JN J VOLUME 4 · NO. 2 nine species: R (Houbraken R (syn. emersonii), and Tanney & Seifert
Table 1. Strains used for phylogenetic analyses of Rasamsonia pulvericola (T £ 6? Name Strain no. Origin GenBank accession no. ITS BenA Cmd R. aegroticola @ #*;'T ¤NN$'' # JX273020.1 ¤NN$K # > < ARTICLE DTO 137B7 ¤NN$'J # JX273012.1 ¤NN$P' # > < R. argillacea /x" #J# $T " > " _ JF417491.1 ¤ R. brevistipitata /x" #N''KT Indoor environment of school, from cork with ¤ Rasamsonia emersonii produces an array of thermostable, MATERIALS AND METHODS lignocellulolytic enzyme cocktails that function at temperatures #J NJ / G Isolation, cultural, and morphological Trichoderma species (Viikari et al. 2007). Cellulase (Moloney characterisation et al. #$'* 6 @ et al. 1993), amylase (Bunni et Settled dust was collected from nine buildings in Thailand al #$'$ !? 4 et al NJJ# !? in April 2009, using sterilised collectors attached to vacuum (Murray et al. 2004) systems have been characterised in ; ? ? 6 / R. emersonii. Potential applications of thermostable enzymes et al. (2007) (Seifert et al., unpubl.) was used to isolate and derived from R. emersonii include food processing, biofuel, 6 and bioconversion processes (Fernandes et al. NJJ' 4;> NJ x 6 + et al. 2010). " 4> #K 4 / ` |¤> # A large-scale survey of the mycobiota of the built L distilled water) and a reduced water activity medium, 20 % ' #P ; et 4; NJ"4;> 4; NJJ 4 al. 2010) resulted in the discovery of a new Rasamsonia $? species, described here as R. pulvericola. This novel Isolates were characterised using morphological species is described and its phylogenetic delimitation and characters and ITS barcode sequences, and maintained Rasamsonia is supported using the internal N 4 6 ; 4; < transcribed spacer (ITS), partial calmodulin (Cmd), and three-point inoculations using conidia suspended in 0.1 % !? BenA). ;4 NPNP*K NPNP* were made on Czapek yeast autolysate agar (CYA), MEA, 206 IMA FUNGUS Rasamsonia pulvericola sp. nov., isolated from house dust Oatmeal Agar (OA), creatine sucrose agar (CREA), 20 % RESULTS ARTICLE / ; / NJ" 6 ? " 6 ~; x R 6 " 6 Rasamsonia strains were isolated on 20SMEA among agar (BLK) (Frisvad & Samson 2004). Colony colours were 907 fungal cultures originating from the Thailand house dust described using the alphanumeric codes of Kornerup & samples. All isolates were recovered from pooled samples #$' @ from one house in Saba Yoi, Songkhla. Morphologically, the ;4 NPNP*K NPNP* fungus is most similar to R. brevistipitata, and is described 4; K / as R. pulvericola below, using the standardised format KMPJ / established by Houbraken et al. (2012). on each medium and temperature treatment in the dark. ITS barcodes and partial Cmd and BenA sequences were Colony diameters and morphological and colony features combined from 22 strains, including all other known species were recorded every 7 d for 1 mo. Microscopic observations of Rasamsonia (Table 1), to investigate the phylogenetic 'K relationship of R. pulvericola in the genus. The data set and an Olympus BX50 light microscope, with micrographs # #$ captured using an Evolution MP Colour Camera (Media for the ITS (559 bases), BenA (497), and Cmd K* / " " 4 ?~ ~ J @ ? 4 4 @ (Media Cybernetics) software. Microscopic measurements N * `@ÅÅ` are presented as ranges calculated from the mean ± @ standard deviation of each measured value, with outliers distinctiveness of nine previously described species, and in brackets. Colony photographs were taken with a Nikon < # / 6 ~KJJJ | @ R. pulvericola in Rasamsonia, assembled using Adobe Photoshop v. 5.5 (Adobe Systems, with R. brevistipitata as the closest sister species. San Jose, CA). ; PJ '$N Thailand house dust samples by 454-pyrosequencing DNA extraction, sequencing, and phylogenetic (Amend et al. 2010). No ITS sequences identical with that of analysis the isolated strains of R. pulvericola were detected in Thailand ` |; 6 N? ? Rasamsonia @_ <N< DJ#;4$ * pulvericola strains using the Ultraclean Microbial DNA Isolation closely related to R. pulvericola, sharing PP PK' with Kit (MoBio Laboratories, Carlsbad, CA) according to the one pairwise alignment gap, was represented by three ITS R @ @" K ' |; sequences in one Micronesian house dust sample. @"P–ITS5 (White et al #$$J @ 6 ;4 NPNP*K NPNP* TAXONOMY ~ !? sequenced to further study the phylogenetic relationship Rasamsonia pulvericola Tanney & Seifert, sp. nov. of R. pulvericola with other Rasamsonia ` 4 x 4x'JP ~/ (Figs 2–3) Houbraken et al. (2012). Newly generated sequences were ` x @ # Etymology: From the Latin pulveris, of dust, -cola, living in, The sequences of the putative new species, along with named for the substrate in which it was discovered, house ` x @ # dust. aligned by Muscle (Edgar 2004) and manually edited and ` ~ K K x ; DiagnosisI ~ > @ ? #JM #P KMN M*' × (1.5–)2–2.5 MrModelTest v. 2.2 (Nylander 2004). Phylogenetic analysis M* ) 4 KM#J K M#* × 2–2.5(–3) was conducted using a Bayesian inference (BI) analysis with ) ~ M KM#J J M#N × N J MN K ) MrBayes v. 3.2 (Ronquist et al. 2012). Four simultaneous of 3–9. Conidia subglobose, smooth walled, pale brown, 2.0– Markov chains were run for 1 000 000 generations (standard 2.5 × N JMN K ) deviation of split frequencies <0.01) with a burn-in time of 250 000 generations and a sampling frequency of 1 in 100 Type: Thailand: Songkhla, Saba Yoi, isolated from settled generations. * # ! |+ NJ$' dust from a single residence, April 2009, P. Noonim (DAOM outgroup for the analysis. NPNP*K M > ;4 NPNK*# M 6? < @" culture). PKP `" <+ PKP + Sciences, Branford, CT) by Amend et al. (2010). The data Description: CulturesI / *J /I were reformatted into a local BLAST database and queried ~; PMK> " *MP> ; NM*> 4; N?*> / NJ" NM*> x+ #> for sequences of our putative new species using BioEdit v. / ;> #> /; Æ# > 7.1.3.0 (Hall 1999). #P ~; #PM#> x+ ##M#*> 4; #JM#N> ; $> / NJ" M'> " M'> / ; NMP> /; # ` NJM*K / VOLUME 4 · NO. 2 207 Tanney & Seifert CBS 101.69T R. argillacea CBS 907.70 R. argillacea 56 DTO 137B7 R. aegroticola 92 DTO 137A8T R. aegroticola A CBS 187.90 R. piperina ARTICLE CBS 408.73T R. piperina CBS 100538T R. eburnea CBS 124447 R. eburnea CGMCC 3.13669T R. composticola 59 CGMCC 3.14946 R. composticola CBS 393.64T R. emersonii CBS 397.64 R. emersonii B CBS 413.71T R. byssochlamydoides 59 99 CBS 533.71 R. byssochlamydoides CBS 275.58T R. cylindrospora CBS 432.62 R. cylindrospora CBS 128785T R. brevistipitata CBS 128786 R. brevistipitata C DAOM 242435T R. pulvericola DAOM 242436 R. pulvericola CBS 103.73 Trichocoma paradoxa NRRL 2098T Talaromyces flavus Fig. 1. / #K JJN x @" !? alignment. Strain accession numbers are followed by the species name (T £ 6? @ # J> # J @ * # ! |+ NJ$' / 6 labelled on the right. *J / NJ / ~; 6 6 *K / smooth entire margin, moderate sporulation, greenish #;M#xI ` Colonies *J / ; \ #/*M#/KI ` ` > #;I ~ /; \ KI x #;PI + 6 N;NI ~ > soluble pigments were absent for all media. colonies on CYA irregular with raised aerial mycelium Conidiophores predominantly arising from aerial in centre, lobate margin, no or poor sporulation, white to > yellowish white (1A2: Yellowish White), reverse white to > #JM K;NI > " #P KMN M*' × # KM NMN K M* ) n = 35). Metulae, when abundant woolly aerial mycelium, lobate margin, moderate present, 7.5–10.5(–13) × NMN K M* ) n = 30), in terminal sporulation, yellowish to pale yellow (1A2–1A3: Pale Yellow), NM* M N KM* K ) K/KI @ G > / NJ" Phialides (7–)7.5–10.0(–12) × N J MN K ) n = 55), in morphologically similar to those on YES but more restricted verticils of 3–9, cylindrical base gradually tapering to a long diameter growth and reverse yellowish green (3B4: Straw collulum (2–4 ) Conidia subglobose, smooth walled, pale > x+ 6 brown, 2.0–2.5 × N JMN K ) n £ J smooth entire margin, moderate to good sporulation, Ascomata or sclerotia not observed. #;M#xI ` moderately concentric with white, light yellow (3A5: Light Notes: Rasamsonia pulvericola is phylogenetically closest *I > to R. brevistipitata. These two species share distinguishing 4; \ characters, including subglobose or ellipsoidal conidia, #;M#xI conidiophores that are usually biverticillate and never ` */KI > 6 208 IMA FUNGUS Rasamsonia pulvericola sp. nov., isolated from house dust ARTICLE Fig. 2. Colonies of Rasamsonia pulvericola (DAOM 242435) #P NK / < I ~; x+ 4; ;> ~; x+ 4; ;> / NJ" " / ; /;> / NJ" YES, CYA, and CREA. compared with other species. The inability of R. pulvericola growth characters. Both species have lower optimum and PJ / 6 Rasamsonia Rasamsonia species. > 6 for R. pulvericola *J / *K / ** / PK / R. brevistipitata. Neither DISCUSSION R. pulvericola nor R. brevistipitata produce the cylindrical conidia typical of other species of the genus. Rasamsonia The recognition of Rasamsonia pulvericola as a pulvericola has a relatively narrow temperature growth novel Rasamonia species was strongly supported by NJM*K / \ morphological characters and molecular phylogenetic @ PJ / analyses. Ramsasonia pulvericola and R. brevistipitata form *K / R. pulvericola a small, distinct clade sharing distinctive morphological and Rasamsonia species. VOLUME 4 · NO. 2 209 Tanney & Seifert ARTICLE Fig. 3. Conidiophores (A–C) and conidia (D–E) of Rasamsonia pulvericola (DAOM 242435) #P NK / 4; x £ #J ) There are minor differences in the phylogram topology was sister to the remaining species in the genus. In contrast, presented here from previous analyses. Su & Cai (2012) the present analysis supports the recognition of three clades found, with low posterior probability, that R. cylindrospora within Rasamsonia, with R. cylindrospora occurring in clade 210 IMA FUNGUS Rasamsonia pulvericola sp. nov., isolated from house dust B (Fig. 1). Rasamsonia pulvericola and R. brevistipitata form Talaromyces emersonii /x" '#P J Enzyme and Microbial ARTICLE the well supported (PPV = 0.99) clade C that is the basal to Technology 11: 370–375. the other two clades. / ¤ ~ ` ~ x x `< NJJ ? Rasamsonia pulvericola was not detected in the ? ? 6 454-pyrosequencing data from Thailand or other countries technique. FEMS Microbiology Ecology 60: 521–533. sampled (Amend et al. 2010). However, one OTU was closely / ` #$P Thermophilic Fungi: an account of related to R. pulvericola (97 %), and probably represents an ' # ! . San Francisco: W.H. additional novel Rasamsonia species in the R. pulvericolaL Freeman. R. brevistipitata clade. This OTU was found in settled dust / D #$P @ 4 Contributions from Boyce Thompson Institute 22: 291–301. * $ / De Ravin SS, Challipalli M, Anderson V, Shea YR, Marciano B, Hilligoss @_ 'JK D, Marquesen M, Decastro R, Liu YC, Sutton DA, Wickes BL, derived from Micronesian dust (Tanney, unpubl. data). Kammeyer PL, Sigler L, Sullivan K, Kang EM, Malech HL, Holland @ ? ? 6 SM, Zelazny AM (2011) Geosmithia argillacea: an emerging cause this rare fungus from the Thailand house dust sample, despite of invasive mycosis in human chronic granulomatous disease. being undetected using the 454-pyrosequencing method. Clinical Infectious Diseases 52I #*M#P* This emphasises the complementarity of using both culture- < " 4 ~` @ 4` NJJ' G ? the thermophilic fungus Talaromyces emersonii for sugar beet microbial biodiversity in environmental samples. The culturing bioconversion. BioResources 3I '$'M$J$ method used to obtain these cultures is labour-intensive Edgar RC (2004) MUSCLE: multiple sequence alignment with high and time-consuming. However, cultures are necessary accuracy and high throughput. Nucleic Acids Research 32: 6 ? 1792–1797. phylogenetic analyses, and allow morphological, metabolic, Flannigan B, Samson RA, Miller JD (eds) (2011) Microorganisms in and enzymatic characterisation. In the absence of cultures, Home and Indoor Work Environments: diversity, health impacts, one might assume R. pulvericola was thermotolerant or investigation and control. 2nd edn. Boca Raton: CRC Press. thermophilic, like other members of the genus. Its mesophilic < ¤/ " ; NJJP ~ 6 Penicillium nature may be surprising for a fungus isolated from a tropical subgenus Penicillium ; ? habitat, but also suggests that comparison of the structure borne terverticillate Penicillia 6 Studies in and function of the enzymes of R. pulvericola with those of its Mycology 49: 1–173. siblings may provide valuable information. The need to populate ` " ~ 4 G x / ¤ | 4 ` x L, Favennec L, Dannaoui E, Bouchara JP, Calenda A (2010) unrepresented but described species is apparent. Such efforts Geosmithia argillacea: an emerging pathogen in patients with 6 Journal of Clinical Microbiology 48I N*'#MN*' data by allowing users to associate sequences with meaningful ` " NJJ* names and corresponding biological information. thermostable enzymes: a review. Bioresource Technology 89: 17–34. Hall TA (1999) Bioedit: a user-friendly biological sequence alignment ACKNOWLEDGEMENTS $KL$'L Nucleic Acids Symposium Series 41I $KM$' We are grateful to Paramee Noonim for collecting the house dust ¤ ` " 4 4 x " < ¤/ 4 ¤< and Jos Houbraken for initially verifying that our ITS sequences x ¤~ " ; NJ#* @ 6 represented a novel Rasamsonia species. Hai Nguyen prepared susceptibility of clinically important Rasamsonia species. Journal ` x ¤ | @ PKP of Clinical Microbiology 51: 22–30. data. This work was supported by a grant to the Indoor Mycobiota Houbraken J, Spierenburg H, Frisvad JC (2012) Rasamsonia, a new Barcode of Life initiative from the Alfred P. 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