Molecular taxonomy of the Alternaria and Ulocladium ... - CBS - KNAW
Molecular taxonomy of the Alternaria and Ulocladium ... - CBS - KNAW
Molecular taxonomy of the Alternaria and Ulocladium ... - CBS - KNAW
Create successful ePaper yourself
Turn your PDF publications into a flip-book with our unique Google optimized e-Paper software.
260 G. S. de Hoog &R.HorréSpeziesgruppen wurde in einer r<strong>and</strong>omisiertenStudie überprüft. Diese Methode erwies sich alsschnell und kostengünstig und ermöglichte sogardie Identifizierung steriler und meristematischer<strong>Alternaria</strong>-Isolate, die zuvor in die GattungenChmelia und Botryomyces eingeordnet wordenwaren. Die mikroskopische Untersuchung sowieeinige zusätzliche Tests bleiben weiterhin notwendig,um einige humanpathogene <strong>Alternaria</strong>-Speziesaggregatezu identifizieren. Etwa 14% derSequenzdaten, die in der Genbank erhältlichsind, erwiesen sich als Fehlidentifizierungen. A.infectoria ist eine der bedeutsamsten <strong>Alternaria</strong>-Artenaus klinischem Material, die jedoch <strong>of</strong>t nurgeringfügig pigmentiert ist. Die fehlende Pigmentierunghat dazu beigetragen, dass entsprechendeIsolate mehrfach falsch identifiziert wurden.IntroductionMembers <strong>of</strong> <strong>the</strong> genus <strong>Alternaria</strong> are among <strong>the</strong>most important airborne agents <strong>of</strong> allergic disorders[1]. In addition, traumatic, localizedinfections are frequently observed [2], which maytake a more severe course in immunocompromisedpatients [3, 4]. In humans, <strong>the</strong> species should beregarded as typical opportunists, <strong>the</strong>ir naturalecological niche being on living or dead plantmaterial.Most infections by <strong>Alternaria</strong> species are attributedto <strong>the</strong> ubiquitous A. alternata (Fr.) Keissl. However,six <strong>Alternaria</strong> species have been reported as potentialagents <strong>of</strong> disease [5]. Morphological distinction <strong>of</strong>species may be difficult due to <strong>the</strong> wide range <strong>of</strong>variability <strong>of</strong> conidia, but some species show clearcutmolecular differences [6, 7]. It was <strong>the</strong> aim <strong>of</strong><strong>the</strong> present study to provide a rapid <strong>and</strong> costeffectivemethodology for <strong>the</strong> distinction <strong>of</strong> clinicallyrelevant entities.We used <strong>the</strong> following research strategy. As areliable starting point was needed prior to development<strong>of</strong> practical diagnostics, <strong>the</strong> <strong>taxonomy</strong> <strong>of</strong>clinically relevant <strong>Alternaria</strong> species is overviewed.Some <strong>Ulocladium</strong> species were included which aremorphologically similar to <strong>Alternaria</strong> <strong>and</strong> which haveoccasionally been reported from humans or clinicalsamples [5]. In addition to morphology (not shown),<strong>the</strong> rDNA internal transcribed spacer (ITS)domain, which is known to display <strong>the</strong> observeddiversity around <strong>the</strong> level <strong>of</strong> species or speciesaggregates [7], was sequenced. Comparativerestriction maps were constructed from whichminimum sets <strong>of</strong> digestions required to recognize<strong>the</strong> clinically relevant entities were abstracted. Thepredictive value <strong>of</strong> this set was verified in a doubleblindstudy.Materials <strong>and</strong> methodsStrains, culture conditions <strong>and</strong> public domain dataClinical strains representing each <strong>of</strong> <strong>the</strong> names usedin <strong>the</strong> literature were collected <strong>and</strong> supplementedwith ex-type <strong>and</strong> au<strong>the</strong>ntic specimens <strong>and</strong> o<strong>the</strong>rstrains <strong>of</strong> <strong>the</strong> same species from nonhuman sources.Reference strains were taken from <strong>the</strong> collection <strong>of</strong><strong>the</strong> Centraalbureau voor Schimmelcultures(Utrecht, <strong>the</strong> Ne<strong>the</strong>rl<strong>and</strong>s), or were kindly donatedby E.G. Simmons (Amherst, MA, USA). When noau<strong>the</strong>ntic live material was available, strainsregarded as representative for <strong>the</strong> species bytaxonomic specialists were taken as such. Strainsstudied (Table 1) were grown on malt extract agar(MEA) <strong>and</strong> potato carrot agar (PCA). Datagenerated were supplemented by all identical <strong>and</strong>closely similar sequences from EMBL ⁄ GenBank.Strains finally clustering as <strong>Alternaria</strong> alternata <strong>and</strong>A. infectoria Simmons were grown on potato dextroseagar (PDA), dichloran Rose Bengal yeast extractsucrose agar (DRYES) <strong>and</strong> Müller–Hinton Agar II(MHA) <strong>and</strong> <strong>the</strong>ir cultural characteristics <strong>and</strong> grossconidiation were recorded. Recipes <strong>of</strong> 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 <strong>of</strong> silica gel <strong>and</strong> 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 <strong>of</strong>200 ll CTAB-buffer <strong>and</strong> vigorous shaking, <strong>the</strong>sample was incubated for 10 min in a 65 °C waterbath. Then, 500 ll chlor<strong>of</strong>orm was added <strong>and</strong> <strong>the</strong>mixture was vortexed briefly <strong>and</strong> centrifuged for5 min at 20 800 g. After <strong>the</strong> aqueous supernatantwas transferred to a new Eppendorf tube, twovolumes (800 ll) ethanol 96%, )20 °C wereadded <strong>and</strong> 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 <strong>and</strong> 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 <strong>and</strong> cooled slowly to room temperature;<strong>the</strong> pH was adjusted to 7.4 by adding 100 ll Tris–HCl). Samples were incubated for 5–30 min at37 °C <strong>and</strong> <strong>the</strong>n stored in a refrigerator.mycoses 45, 259–276 (2002)