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

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264 G. S. de Hoog &R.HorréTable 2.Misidentified strainsNumber Current name Correct identificationNCBI AF218791 Alternaria alternata Ulocladium chartarumCBS 198.74 Alternaria chlamydospora unidentified speciesNCBI D38768 Alternaria dauci Alternaria porriNCBI D38760 Alternaria porri unidentified speciesNCBI AF307015 Alternaria radicina Alternaria petroseliniNCBI U80204 Alternaria solani unidentified speciesNCBI AF133843 Cylindrocarpon lichenicola Alternaria alternataCBS 103.47 Ulocladium botrytis Ulocladium atrumCBS 104.21 Ulocladium botrytis Ulocladium atrumNCBI AF229487 Ulocladium botrytis Ulocladium atrumCBS 452.72 Ulocladium botrytis Ulocladium atrumCBS 453.74 Ulocladium mouchaccae unidentified speciesTotal number of misidentified strains: GenBank: seven out of 49; CBS: five out of 50.eluted according to protocols given by the supplier,and collected with TE-buffer. Concentrations ofamplicons were estimated by comparison withSmartLadder markers (Eurogentec, Seraing, Belgium)on 1% agarose gels. Sequencing primers wereITS1, ITS4 and ITS5; reactions (96°C for 10 s;50°C for 5 s; 60°C for 4 min; 25 cycles) werecarried out with 15–50 ng of DNA for a 10-llreaction mixture including 4 pmol primer and 4 llBigDye RR Mix (Applied Biosystems, Nieuwerkerka ⁄ d IJssel, the Netherlands). Subsequently, DNAwas precipitated with ethanol and sequenced usingan ABI Prism 310 Genetic Analyzer (AppliedBiosystems).Alignment and phylogenetic analysisSequences were adjusted using the SeqManprogram within the Lasergene software package(DNASTAR, Madison, WI, USA) and alignedusing BioNumerics (Applied Maths, Kortrijk,Belgium). A distance tree was constructed usingneighbor-joining with a Kimura correction usingthe Treecon (version 1.3b) software package [10],and a phylogenetic tree was constructed using paupversion 4.0b8 with heuristic search option andstepwise addition. Bootstrap values were calculatedfrom 100 resampled datasets.ResultsIdentity of strainsFor each strain included in the tree, an authority forits identification is given (Table 1). Ex-type materialsare also indicated in the Table. Authorities areexperts in the field who confirmed the identity ofthe strain. A reference to a taxonomic publicationciting the strain is given where appropriate. Correctsequence identity can be judged from the alignmentof rDNA ITS sequences using the Ward’s averagingoption in BioNumerics. Conclusions based on thesedata are supplemented with morphology andliterature data. The reference strains provide areliable framework for the phylogenetic tree.Misidentified strains are listed in Table 2. InGenBank, 14% of the sequences held prove to beincorrectly identified. A marked example is Cylindrocarponlichenicola (Massal.) Hawksworth, AF133843. The species is remote from Alternaria,belonging to the order Hypocreales rather thanPleosporales, but its sequence appears to beidentical to that of A. alternata. The authorsprobably sequenced a contaminant. In the CBSculture collection, made up of strains primarilyTable 3. Species having ITS1–2 rDNA sequences nearlyidentical to the species printed in bold typeAlternaria alternataAlternaria arborescensAlternaria liniAlternaria longipesAlternaria maliAlternaria tenuissimaAlternaria radicinaAlternaria carotiincultaeAlternaria petroseliniAlternaria seliniAlternaria raphaniAlternaria japonicaAlternaria solaniAlternaria linicolaUlocladium atrumUlocladium dauciUlocladium multiformeUlocladium cucurbitaeUlocladium botrytisUlocladium alternariaemycoses 45, 259–276 (2002)
Alternaria and Ulocladium molecular taxonomy 265Table 4.Restriction enzymes usedTable 5.Overview of restriction sitesEnzymeAccIIDdeIHaeIIIHhaIHinf IMspIRsaISau3A1TaqIRecognition siteCG ⁄ CGC⁄ TNAGGG ⁄ CCGCG ⁄ CG⁄ ANTCC⁄ CGGGT⁄ AC⁄ GATCT⁄ CGAidentified by morphology, 10% of the Alternariastrains have been misidentified (Table 2). Somespecies have nearly identical ITS sequences(Table 3). They are mostly also morphologicallysimilar and are frequently found on the same hostplant. Information on whether these are separatespecies was not gained in the course of this study.Cultural characteristicsCBS strains assigned after sequencing to theAlternaria alternata and Lewia infectoria clusters grewpoorly on media with high sugar concentration(Table 7), being highly degenerative on DRYESmedium. On this medium cultures were invariablysterile, with white, dense, cushion-shaped aerialmycelium, sometimes deeply furrowed or wrinkledand then of hard texture and olivaceous brown. Noconsistent difference between the two species wasnoted in growth on the media.ITS sequencingStrains of Alternaria infectoria ⁄ Botryomyces caespitosus deHoog & Rubio [11] (Fig. 1) (group 1) were found tocompose a clade differing clearly from theremaining species. The ITS of strains contain anindel of about 30 bp starting at position 23 of ITS1.An insertion of similar length but with differentbase composition is found at the same position in agroup of four strains containing Alternaria mouchaccaeSimmons including the ex-type strain, CBS 490.72(group 2). The latter indel is also present in strainUlocladium botrytis Preuss, CBS 197.67, albeit aminor 4 bp difference. All remaining Alternaria andUlocladium strains analyzed lack an insertion at thisposition. The cluster A. infectoria ⁄ B. caespitosus (1) issplit at low resolution by differences at twophylogenetically informative sites (i.e. recurrent indifferent strains). Botryomyces caespitosus also differsfrom A. infectoria by an additional 10 unique bases(i.e. found in a single strain only).A core group (group 3) of 31 strains, includingCBS 603.78 and CBS 916.96 are regarded by E.G.Strains with ITS domains of about 600 bp:ITS1: U. botrytis B. caespitosus A. infectoriaHinf I 57 – –HhaI 92 85 90AccII – 85 91HaeIII – 93 99MspI – 94 100HaeIII 101 97 103RsaI 138 – 136DdeI – 162 1685.8S:Sau3A1 246 239 234TaqI 265 257 263Hinf I 318 310 316TaqI 324 316 322Hinf I 326 318 324HhaI 347 339 345TaqI 377 369 375ITS2:RsaI 391 383 389Hinf I 441 – 441MspI 465 459 465HaeIII 468 462 468HhaI 486 – 486AccII 498 492 498HhaI 500 494 500Backward 603 598 605primerStrains with ITS domains of about 570 bp:A. alternata A. clamydospora U. atrum U. chartarumITS1:Hinf I – – – 57RsaI 109 111 108 1085.8S:Sau3A1 215 219 215 216TaqI 233 239 234 235Hinf I – 292 287 288TaqI 293 298 293 293HhaI 317 321 316 317Hinf I 296 300 295 296TaqI 347 351 346 347ITS2:RsaI 362 365 360 361Hinf I 413 415 410 411MspI 437 439 434 435HaeIII 440 442 437 438HhaI 458 460 455 456AccII – – 467 468HhaI – – 469 469Backward 578 578 572 573primerSites usable for diagnostics (criteria see text) written in boldtype.Simmons as strains representative for Alternariaalternata. Seven of these strains are strictly identical.Among them is CBS 101.13, ex-type strain ofAlternaria geophila Dawszewska, an establishedsynonym of A. alternata, as well as four strainsdeposited under the name Alternaria tenuissima(Kunze:Pers.) Wiltsh., for which no type materialis known to be preserved. Group 3, with CBSmycoses 45, 259–276 (2002)
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