J. Phytopathology 144, 347-354 (1996) © 1996 Blackwell Wissenschafts-Verlag, Berlin ISSN 0931-1785 In.stitute of Applied Microbiology, University of Agriculture, Nussdorfer Lande Jl, Vienna, Austria Molecular Characterization of the Plant Pathogen Verticillium dahliae Kleb. Using RAPD-PCR and Sequencing of the 18SrRNA-Gene R. MESSNER'. W. SCHWEIGKOFLER', M. IBL'. G. BERG^ and H. PRILLINGER' 'Institute of Applied Microbiology. University of Agriculture, Nussdorfer Lande 11. Vienna, Austria; 'Codon Genetic Systems. A-l 180 Vienna, Austria; 'Fachbereich Biologie, University of Rostock, D-18051 Rostock, Germany (correspondence to H. Prilhnger) With 4 figures Received November 22, 1995; accepted May 21. 1996 Abstract Thirty-four isolates of Verticillium dahliae Kleb. from nine diflferent genera of dicotyledonous host plants and a broad range of geographic regions were analysed geno- typically. Random amplified polymorphic DNA (RAPD) markers were used for the estimation of the genetic varia- bility within the species. Using four primers for the analy- sis, 79 distinct fragments were obtained. The derived phenogram clustered the isolates in two main groups; one consisted almost entirely of V. dahliae isolates from oilseed rape (Brassica napus napus). the other group com- prised isolates from a wide range of host plants. No correlation between geographic location of the isolates and the RAPD-pattern was observed. Sequencing of the gene for the 18SrRNA and cal- culation of the phylogenetic tree integrated the deut- eromycetous fungus V. dahliae into the sexual system of the filamentous ascomycetes. Zusammenfassung Molekulare Charakterisierung des Pflanzenpathogens VerticilUum dah- liae Kleb. mitteis RAPD-PCR und SeqaeiHiening des I^rRNA-Gens. VierunddreiCig Isolate von Verticillium dahliae Kleb. aus neun verschiedenen dikotylen Wirtspflanzen und unter- schiedlichen geographischen Regionen wurden ge- notypisch analysiert. Zur Bestimmung der genetischen Variabilitat innerhalb der Art wurden "Random- Atnplified-Polymorphic-DNA" (RAPD)-Marker ver- wendet. Durch den Einsatz von vier Primern wurden 79 distinkte Fragmente erhalten, das daratis berechnete Phenogramm gruppiert die Isolate in zwei Cluster; der erste enthalt V. dahliae Isolate aus Raps (Brassica napus napus), der zweite umfaBt Isolate aus einem breiten Wirtsspektrtim. Die geographische Herkunft der Isolate zeigt keine Korrelation zum erhaltenen RAPD-Muster. Sequenziening des Gens fur die 18SrRNA und an- schliessende Stammbaumberechnung integrierte den Deuteromyceten V. dahliae in das sexuelle System der fllamentosen Ascomyceten. Introduction The hyphomycete Verticillium dahliae Kleb. is a root- associated fungus of worldwide distribution and has been cultured from blackleg-affected oilseed rape {Brassica napus napus), paprika (Capsicum annuum), flax (Linum usitatissimum). grape (Vitis vinifera) and a wide range of other plants of economic value in which it causes tracheomycosis (Verticillium-wilt). Host-specific isolates of V. dahliae are indistinguishable on the basis of mor- phological characters. Distinctive characters of Ver- ticillium species within the section Nigresceniia are the formation of dark resting mycelium by V. albo-atrum, of dark microsclerotia by V. dahliae and of resting mycelium, microsclerotia and chlamydospores by V. tricorpus. Unfortunately, members of this genus may loose some of the distinctive characters after prolonged maintenance in culture or when kept on an inappropriate medium. There have been many attempts to separate Ver- ticillium albo-atrum and V. dahliae and other Verticillium species at the species level recently (Carder and Barbara, 1991; Nazar et ah, 1991; Williams et al., 1992; Carder et al., 1993; Li et al., 1994). On the basis of the nucleotide sequences of an amplified mitochondrial smail rRNA gene region Li et al. (1994) developed specific primers that amphfied a 140-bp region of V. dahliae DNA. The V. dahliae-spedfic PCR primer may aid in more rapid and specific detection of the pathogen directly in plant and/or soil samples. Further differentiation of specific pathotypes from V. albo-atrum and V. dahliae was reported from Okoli et al. (1993, 1994) using restriction fragment length polymorphisms (RFLPs). Two distinct groups were shown in V. aibo-atrum: isolates from lucerne formed one group and those from all other hosts the second group, ln V. dahliae four distinct groups were Copyright CtoranceCemer Code Sta,em=«: 0931^1785/96/4408-0347 S 11.50/0