Molecular taxonomy of the Alternaria and Ulocladium ... - CBS - KNAW

260 G. S. de Hoog &R.HorréSpeziesgruppen wurde in einer randomisiertenStudie überprüft. Diese Methode erwies sich alsschnell und kostengünstig und ermöglichte sogardie Identifizierung steriler und meristematischerAlternaria-Isolate, die zuvor in die GattungenChmelia und Botryomyces eingeordnet wordenwaren. Die mikroskopische Untersuchung sowieeinige zusätzliche Tests bleiben weiterhin notwendig,um einige humanpathogene Alternaria-Speziesaggregatezu identifizieren. Etwa 14% derSequenzdaten, die in der Genbank erhältlichsind, erwiesen sich als Fehlidentifizierungen. A.infectoria ist eine der bedeutsamsten Alternaria-Artenaus klinischem Material, die jedoch oft nurgeringfügig pigmentiert ist. Die fehlende Pigmentierunghat dazu beigetragen, dass entsprechendeIsolate mehrfach falsch identifiziert wurden.IntroductionMembers of the genus Alternaria are among themost important airborne agents of allergic disorders[1]. In addition, traumatic, localizedinfections are frequently observed [2], which maytake a more severe course in immunocompromisedpatients [3, 4]. In humans, the species should beregarded as typical opportunists, their naturalecological niche being on living or dead plantmaterial.Most infections by Alternaria species are attributedto the ubiquitous A. alternata (Fr.) Keissl. However,six Alternaria species have been reported as potentialagents of disease [5]. Morphological distinction ofspecies may be difficult due to the wide range ofvariability of conidia, but some species show clearcutmolecular differences [6, 7]. It was the aim ofthe present study to provide a rapid and costeffectivemethodology for the distinction of clinicallyrelevant entities.We used the following research strategy. As areliable starting point was needed prior to developmentof practical diagnostics, the taxonomy ofclinically relevant Alternaria species is overviewed.Some Ulocladium species were included which aremorphologically similar to Alternaria and which haveoccasionally been reported from humans or clinicalsamples [5]. In addition to morphology (not shown),the rDNA internal transcribed spacer (ITS)domain, which is known to display the observeddiversity around the level of species or speciesaggregates [7], was sequenced. Comparativerestriction maps were constructed from whichminimum sets of digestions required to recognizethe clinically relevant entities were abstracted. Thepredictive value of this set was verified in a doubleblindstudy.Materials and methodsStrains, culture conditions and public domain dataClinical strains representing each of the names usedin the literature were collected and supplementedwith ex-type and authentic specimens and otherstrains of the same species from nonhuman sources.Reference strains were taken from the collection ofthe Centraalbureau voor Schimmelcultures(Utrecht, the Netherlands), or were kindly donatedby E.G. Simmons (Amherst, MA, USA). When noauthentic live material was available, strainsregarded as representative for the species bytaxonomic specialists were taken as such. Strainsstudied (Table 1) were grown on malt extract agar(MEA) and potato carrot agar (PCA). Datagenerated were supplemented by all identical andclosely similar sequences from EMBL ⁄ GenBank.Strains finally clustering as Alternaria alternata andA. infectoria Simmons were grown on potato dextroseagar (PDA), dichloran Rose Bengal yeast extractsucrose agar (DRYES) and Müller–Hinton Agar II(MHA) and their cultural characteristics and grossconidiation were recorded. Recipes of media havebeen given by De Hoog et al. [5]; MHA was obtainedfrom Becton Dickinson (Heidelberg, Germany).DNA extractionApproximately 1 g mycelium was transferred to a2 : 1 mixture of silica gel and Celite 545 with 300 llcetyltrimethylammonium bromide (CTAB)-bufferadded (Tris–HCl, 200 mmol l )1 , pH 7.5;Na-EDTA, 200 mmol l )1 ; NaCl 8.2%; CTAB2%). The material was ground with a micropestle(Eppendorf, Hamburg, Germany). After addition of200 ll CTAB-buffer and vigorous shaking, thesample was incubated for 10 min in a 65 °C waterbath. Then, 500 ll chloroform was added and themixture was vortexed briefly and centrifuged for5 min at 20 800 g. After the aqueous supernatantwas transferred to a new Eppendorf tube, twovolumes (800 ll) ethanol 96%, )20 °C wereadded and mixed gently. The DNA was precipitatedat )20 °C for at least 30 min. The pellet, obtainedby centrifugation for 5 min at 20 800 g, was washedtwice with 500 ll ethanol 70% at )20 °C. DNA wasdried overnight at room temperature and suspendedin 97.5 ll TE-buffer (10 mmol l )1 Tris, 10 mmNa-EDTA, pH 8.0) with 2.5 ll RNAse-solution(10 mg pancreatic RNase 20 U mg )1 was added to1 ml 0.01 mol l )1 sodium acetate, heated at 100 °Cfor 15 min and cooled slowly to room temperature;the pH was adjusted to 7.4 by adding 100 ll Tris–HCl). Samples were incubated for 5–30 min at37 °C and then stored in a refrigerator.mycoses 45, 259–276 (2002)