mycoses 45, 259–276 (2002) Accepted:November 5, 2001 Molecular taxonomy of the Alternaria and Ulocladium species from humans and their identification in the routine laboratory Molekulare Taxonomie humaner Isolate von Alternaria- und Ulocladium-Arten und ihre Identifizierung im Routinelabor G. S. de Hoog1 and R. Horre´2 Keywords. Alternaria, Ulocladium, melanized fungi, opportunistic fungi, rDNA, ITS-sequencing, RFLP, identification. Schlu¨ sselwo¨rter. Alternaria, Ulocladium, Schwa¨rzepilze, Opportunisten, rDNA, ITS-Sequenzierung, RFLP, Identifizierung. Summary. The Alternaria and Ulocladium species remain necessary to allow identification of the reported from humans are studied taxonomically aggregates of potential etiological agents of human using rDNA internal transcribed spacer (ITS) disease. About 14% of the sequences deposited in sequence data. The ITS variability within the GenBank were found to be misidentified. Alternaria genus is relatively limited. The two most important, infectoria is one of the most common clinical longicatenate species, Alternaria alternata and Alternaria species, despite its low degree of melani- A. infectoria, clearly differ in their ITS domains, zation. The lack of pigmentation has frequently led due to a 26-bp insert in ITS1 of the latter species. A to misidentification of such isolates. number of taxa inhabiting particular plant species, such as A. longipes on tobacco and A. mali on apple, Zusammenfassung. Die Alternaria- und Ulo- but also the common saprobic species A. tenuissima cladium-Spezies, die als klinische Isolate beschrie- cannot reliably be distinguished from A. alternata ben worden sind, wurden taxonomisch mittels using this method. The large number of described rDNA ITS (Internal Transcribed Spacer-) noncatenate, obligatory plant pathogens are Sequenzanalysen untersucht. Die ITS-Variabilita¨t extremely rare as agents of human disease; clinical innerhalb dieser Gattungen ist relativ begrenzt. routine identification does not need to include these Die beiden humanmedizinisch bedeutsamsten taxa. The predictivity of a simplified polymerase Arten, Alternaria alternata und A. infectoria, chain reaction–restriction fragment length poly- unterscheiden sich eindeutig voneinander in ihrer morphism procedure of rDNA for the recognition ITS-Doma¨ne, vor allem durch eine Insertion in of the relevant species or species aggregates is ITS1 von 26 Bp bei der letztgenannten Spezies. established in a randomized test. The method was Manche pflanzenpathogene Arten, wie beispiels- found to be rapid and cost-effective. Its efficacy weise A. longipes auf Tabakpflanzen und A. mali extended to the identification of sterile and meris- auf A¨ pfeln, sowie die ha¨ufig isolierte saprobische tematic Alternaria strains, some of them previously Art A. tenuissima,ko¨nnen mit dieser Methode classified in genera such as Chmelia and Botryomyces. nicht eindeutig von A. alternata unterschieden Microscopic morphology and some additional tests werden. Die meisten der pflanzenpathogenen Arten ohne Konidienketten werden jedoch extrem selten als Verursacher humaner Mykosen beo- 1Centraalbureau voor Schimmelcultures (CBS), Utrecht, the Netherlands, and 2Institut fu¨r Medizinische Mikrobiologie, bachtet, weshalb sie klinisch kaum relevant sind Universita¨t Bonn, Bonn, Germany. und ihre Identifizierung in Routinelaboratorien als nicht erforderlich erachtet werden kann. Die Correspondence: Prof. Dr G. S. de Hoog, Centraalbureau voor Identifizierungsfa¨higkeit einer vereinfachten PCR- Schimmelcultures, PO Box 85167, NL-3508 AD Utrecht, the Netherlands. Tel.: +31–30–2122663 Fax: +31–30–2512097 RFLP-Analyse der rDNA zur Bestimmung der E-mail: [email protected] humanmedizinisch bedeutsamen Spezies oder 260 G. S. de Hoog &R.Horre´ Speziesgruppen wurde in einer randomisierten Materials and methods Studie u¨berpru¨ft. Diese Methode erwies sich als schnell und kostengu¨nstig und ermo¨glichte sogar Strains, culture conditions and public domain data die Identifizierung steriler und meristematischer Alternaria-Isolate, die zuvor in die Gattungen Clinical strains representing each of the names used Chmelia und Botryomyces eingeordnet worden in the literature were collected and supplemented waren. Die mikroskopische Untersuchung sowie with ex-type and authentic specimens and other einige zusa¨tzliche Tests bleiben weiterhin notwen- strains of the same species from nonhuman sources. dig, um einige humanpathogene Alternaria-Spezies- Reference strains were taken from the collection of aggregate zu identifizieren. Etwa 14% der the Centraalbureau voor Schimmelcultures Sequenzdaten, die in der Genbank erha¨ltlich (Utrecht, the Netherlands), or were kindly donated sind, erwiesen sich als Fehlidentifizierungen. A. by E.G. Simmons (Amherst, MA, USA). When no infectoria ist eine der bedeutsamsten Alternaria-Arten authentic live material was available, strains aus klinischem Material, die jedoch oft nur regarded as representative for the species by geringfu¨gig pigmentiert ist. Die fehlende Pigmen- taxonomic specialists were taken as such. Strains tierung hat dazu beigetragen, dass entsprechende studied (Table 1) were grown on malt extract agar Isolate mehrfach falsch identifiziert wurden. (MEA) and potato carrot agar (PCA). Data generated were supplemented by all identical and closely similar sequences from EMBL ⁄ GenBank. Introduction Strains finally clustering as Alternaria alternata and A. infectoria Simmons were grown on potato dextrose Members of the genus Alternaria are among the agar (PDA), dichloran Rose Bengal yeast extract most important airborne agents of allergic dis- sucrose agar (DRYES) and Mu¨ller–Hinton Agar II orders [1]. In addition, traumatic, localized (MHA) and their cultural characteristics and gross infections are frequently observed [2], which may conidiation were recorded. Recipes of media have take a more severe course in immunocompromised been given by De Hoog et al. [5]; MHA was obtained patients [3, 4]. In humans, the species should be from Becton Dickinson (Heidelberg, Germany). regarded as typical opportunists, their natural ecological niche being on living or dead plant DNA extraction material. Most infections by Alternaria species are attributed Approximately 1 g mycelium was transferred to a to the ubiquitous A. alternata (Fr.) Keissl. However, 2 : 1 mixture of silica gel and Celite 545 with 300 ll six Alternaria species have been reported as potential cetyltrimethylammonium bromide (CTAB)-buffer ) agents of disease [5]. Morphological distinction of added (Tris–HCl, 200 mmol l 1, pH 7.5; ) species may be difficult due to the wide range of Na-EDTA, 200 mmol l 1; NaCl 8.2%; CTAB variability of conidia, but some species show clear- 2%). The material was ground with a micropestle cut molecular differences [6, 7]. It was the aim of (Eppendorf, Hamburg, Germany). After addition of the present study to provide a rapid and cost- 200 ll CTAB-buffer and vigorous shaking, the effective methodology for the distinction of clini- sample was incubated for 10 min in a 65 °C water cally relevant entities. bath. Then, 500 ll chloroform was added and the We used the following research strategy. As a mixture was vortexed briefly and centrifuged for reliable starting point was needed prior to devel- 5 min at 20 800 g. After the aqueous supernatant opment of practical diagnostics, the taxonomy of was transferred to a new Eppendorf tube, two clinically relevant Alternaria species is overviewed. volumes (800 ll) ethanol 96%, )20 °C were Some Ulocladium species were included which are added and mixed gently. The DNA was precipitated morphologically similar to Alternaria and which have at )20 °C for at least 30 min. The pellet, obtained occasionally been reported from humans or clinical by centrifugation for 5 min at 20 800 g, was washed samples [5]. In addition to morphology (not shown), twice with 500 ll ethanol 70% at )20 °C. DNA was the rDNA internal transcribed spacer (ITS) dried overnight at room temperature and suspended ) domain, which is known to display the observed in 97.5 ll TE-buffer (10 mmol l 1 Tris, 10 mm diversity around the level of species or species Na-EDTA, pH 8.0) with 2.5 ll RNAse-solution ) aggregates [7], was sequenced. Comparative (10 mg pancreatic RNase 20 U mg 1 was added to ) restriction maps were constructed from which 1 ml 0.01 mol l 1 sodium acetate, heated at 100 °C minimum sets of digestions required to recognize for 15 min and cooled slowly to room temperature; the clinically relevant entities were abstracted. The the pH was adjusted to 7.4 by adding 100 ll Tris– predictive value of this set was verified in a double- HCl). Samples were incubated for 5–30 min at blind study. 37 °C and then stored in a refrigerator. mycoses 45, 259–276 (2002) Alternaria and Ulocladium molecular taxonomy 261 Table 1. List of strains and sequences studied with collection and GenBank, EMBL and DDBJ numbers Name Numbers Source Geography Isolated by Authority A. alternata AA6 ¼ U05195 Brassica rapa spp. Canada G.A. Petrie [27] oleifera A. alternata CBS 105.49 contaminant blood Italy M. Poli – culture A. alternata CBS 101.13, T for soil Switzerland W. Daszewska [58] A. geophila A. alternata CBS 103.33 soil Egypt F.H. van Beyma – A. alternata CBS 108.41 ¼ ATCC wood – S. Truter – 11892 A. alternata IFO 4026 ¼ D38756 – Japan T. Hasegawa – + D38764 A. alternata CBS 603.78 ¼ QM 9553, air USA, Texas G.H. Meyer E.G. Simmons REPR for A. alternata A. alternata dH 10735 (received as human skin lesion India S.M. Singh [12] A. chlamydospora) case 2 A. alternata