J. Phytopathology 144, 347-354 (1996)© 1996 Blackwell Wissenschafts-Verlag, BerlinISSN 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 GeneticSystems. 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

AbstractThirty-four isolates of Verticillium dahliae Kleb. fromnine diflferent genera of dicotyledonous host plants anda 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 derivedphenogram clustered the isolates in two main groups;one consisted almost entirely of V. dahliae isolates fromoilseed rape (Brassica napus napus). the other group com-prised isolates from a wide range of host plants. Nocorrelation between geographic location of the isolatesand 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 ofthe filamentous ascomycetes.

ZusammenfassungMolekulare Charakterisierung des Pflanzenpathogens VerticilUum dah-

liae Kleb. mitteis RAPD-PCR und SeqaeiHiening des I^rRNA-Gens.

VierunddreiCig Isolate von Verticillium dahliae Kleb. ausneun verschiedenen dikotylen Wirtspflanzen und unter-schiedlichen geographischen Regionen wurden ge-notypisch analysiert. Zur Bestimmung der genetischenVariabilitat innerhalb der Art wurden "Random-Atnplified-Polymorphic-DNA" (RAPD)-Marker ver-wendet. Durch den Einsatz von vier Primern wurden79 distinkte Fragmente erhalten, das daratis berechnetePhenogramm gruppiert die Isolate in zwei Cluster; dererste enthalt V. dahliae Isolate aus Raps (Brassica napusnapus), der zweite umfaBt Isolate aus einem breitenWirtsspektrtim. Die geographische Herkunft der Isolatezeigt 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 derfllamentosen Ascomyceten.

IntroductionThe hyphomycete Verticillium dahliae Kleb. is a root-associated fungus of worldwide distribution and has beencultured from blackleg-affected oilseed rape {Brassicanapus napus), paprika (Capsicum annuum), flax (Linumusitatissimum). grape (Vitis vinifera) and a wide rangeof other plants of economic value in which it causestracheomycosis (Verticillium-wilt). Host-specific isolatesof V. dahliae are indistinguishable on the basis of mor-phological characters. Distinctive characters of Ver-ticillium species within the section Nigresceniia are theformation of dark resting mycelium by V. albo-atrum, ofdark microsclerotia by V. dahliae and of restingmycelium, microsclerotia and chlamydospores by V.tricorpus. Unfortunately, members of this genus mayloose some of the distinctive characters after prolongedmaintenance in culture or when kept on an inappropriatemedium.

There have been many attempts to separate Ver-ticillium albo-atrum and V. dahliae and other Verticilliumspecies at the species level recently (Carder and Barbara,1991; Nazar et ah, 1991; Williams et al., 1992; Carder etal., 1993; Li et al., 1994). On the basis of the nucleotidesequences of an amplified mitochondrial smail rRNAgene region Li et al. (1994) developed specific primersthat amphfied a 140-bp region of V. dahliae DNA. TheV. dahliae-spedfic PCR primer may aid in more rapidand specific detection of the pathogen directly in plantand/or soil samples. Further differentiation of specificpathotypes from V. albo-atrum and V. dahliae wasreported from Okoli et al. (1993, 1994) using restrictionfragment length polymorphisms (RFLPs). Two distinctgroups were shown in V. aibo-atrum: isolates fromlucerne formed one group and those from all other hoststhe second group, ln V. dahliae four distinct groups were

Copyright CtoranceCemer Code Sta,em=«: 0 9 3 1 ^ 1 7 8 5 / 9 6 / 4 4 0 8 - 0 3 4 7 S 11.50/0

348 MESSNER et al.

found: diploid isolates comprised a group (D) only dis-tantly related to either haploid isolates of V, dahliae orto V, albo-atrum. Most V. dahliae-isolates were haploidand fell into three further groups (A, B and C) (Horiuchiet a!., 1990). Additional studies from Carder and Barbara(1994) using isolates from Japan and North Americashowed an even broader range of variation in the RFLP-pattems.

Recent work by Morton et al. (1995a,b) using sub-repeat sequences in the RNA intergenic regions andsequences of the internal transcribed spacers (ITS)showed a remarkable extent of differences between hap-loid and diploid isolates of V. dahliae, may be evenenough to put them into two separate specira.

Attempts to use fungicide-resistance-markers (e.g.Johnson et al., 1994) and virulence markers (e.g.Schreiber and Domir, 1994) that describe genetic vari-ation in plant pathogen populations are obviously impor-tant because they provide direct information concerningthe effects of selection and the potential effectiveness ofresistance genes. However, virulence and resistance mar-kers may represent only a small portion of the totalgenetic variation present in the population. As shown bySamborski and Dyck (1976) for the Puccinia recondita f.sp. tritici-Trilicum aestivum-system, virulence/avirulencewas most often inherited as a single gene that cor-responded to a specific resistance gene. Conclusionsdrawn exclusively on race phenotypes of plant pathogenicfungi which were derived by this gene-for-gene concept(Flor, 1971) may not give sufficient information on thelevel of other genetic differences either between races orbetween independent collections of a race.

In recent years 'scanning' the DNA of isolates at lociarbitrarily selected across the genome using randomamplified polymorphic DNA (RAPD) analysis, hasbecome a widespread approach in plant pathology (e.g.Schafer and Wostemeyer, 1992).

Cluster analysis of resulting bands are able to dis-tinguish between geographical races (e.g. Assigbetse etal., 1994; Kolmer et al,, 1995; Maclean et al., 1995),pathotypes restricted to a sole host plant (e.g. Haydenet al,, 1994) and somatic hybrids following parasexualrecombination in Penicillium roqueforti (Durand et al.,1993).

The clear genotypic differentiation of parasitic fungiinto pathotypes of variable aggressiveness is a pre-requisite for biological plant protection using resistanceinduction in the host plant. For example, elm trees whichwere pre-treated with a less aggressive strain of Ophios-loma novo-ulmi survived a subsequent inoculation with ahighly aggressive type (Scheffer, 1990).

The introduction of deuteromycetes into the system ofascomycetes and basidiomycetes nowadays seems poss-ible using molecular techniques. Using the sequence ofthe gene for the 18S subunit of ribosomal RNA, Berbeeand Taylor (1992a,b) showed, that the anamorphichuman pathogen Sporothrix schenkii lies phylogeneticailywithin the sexual genus Ophiostoma. Rehner and Samuels(1994), working with the nuclear large subunit ribosomalDNA (28SrDNA), showed members of the genus Gli-

odadium to be polyphyletic and generically distinct,although showing great morphological similarities. Theimportance of these findings for the future design ofmolecular plant protection is obvious.

The present study investigated molecular heterogeneityand phylogenetic position of the plant pathogen Ver-ticillium dahliae using RAPD-PCR and sequencing ofthe18SrDNA.

Materials and MethodsFungal strains

The strains examined are listed in Table 1. All strains aremaintained at the culture collection of the Institute ofApplied Microbiology, IAM, University of Agriculture,Vienna; Austria.

DNA extraction

Fungal strains were cultured in 100 ml Erlenmeyer-flaskscontaining 20 ml Mandels-Andreotti-Medium (per liter:10 g glucose; 2 g peptone; 2.8 g ammonium sulfate; 4 gKH,PO4; 10 g Na,HPO4, pH 5.0; 10 ml of a simplifiedCzapek cone: 7 g MgSO^; 0.05 g CUSO4 x 511,0; 0.1 gFeSO* X 7H,O; 0.1 g ZnSOj x 7H,O) for five days usinga rotary shaker (30 C; 150 rpm).

The mycelium was coliected by filtration and groundto a fine powder in liquid N2. Fifty mg of the groundwere transferred to a 1.5 ml Eppendorf tube and mixedwith 0.4 ml lysis buffer (0.1 M Tris; 1.4 M NaCI; 50 mMEDTA; pH 8.0). The suspension was frozen at — 20 Cand thawed three times. After addition of 0.4 m! phenolfreezing and thawing was repeated. The suspension washeated at 55 C for 10 min, 0.4 ml of chloroform wereadded and heated again. The resulting mixture was cen-trifugated at 15 500 x ̂ for 30 min, and 0.3 ml of the clearsupernatant was mixed with 0.45 ml of propan-2-o!. Afterincubation at room temperature for 5 min the mixturewas centrifuged at 15 500 x g for 10 min and the resultingpellet washed twice with 0.8 ml of 70% ethanol. Thepellet was dried under vacuum and dissolved in 50 ii\ TE-buffer. Samples chosen for sequencing were digested with2.5 n\ RNAse (10 mg/ml) for 3 h at 37 C. DNA con-centrations were evaluated by agarose ge! electrophoresis.

RAPD-analysisThe RAPD-PCR was performed as described in Messneret aL (1994), using the primers M13(5dGAGGGTGGCGGTrCT); GACA (5dGA-CAGACAGACAGACA); (both Lieckfeldt et al., 1993);VI (5'dACGGTCTTGG; Schafer and Wostemeyer,1992) and V5 (5'dTGCCGAGCTG; Caetano-Anolles etal., 1992). The temperature profile for primers M13 andGACA was as follows: denaturation at 98°C for 15 s;annealing at 50°C for 60 s and extension at 72°C for 100s for a total of 40 cycles. For decamer primers VI and V5annealing was performed at 32°C for 90 s. Synthesisof primers was performed by Codon Genetic Systems(Vienna; Austria) using a model 392 DNA synthesizer{Applied Biosystems, Foster City, CA, USA).

Computer analysis of RAPD patterns was performedas in Halmschlager et al, (1994), Basically, the infor-

Molecular Characterization of the Plant Pathogen Vertieillium 349

Table 1List of fungal strains studied

Spedes

V. dahUae

lAM no.

MD15MD44MD45MD46MD47MD48MD49MD53MD54MD55MD56MD57MD5SMD59MD68MD69MD70MD71MD73MD74MD75MD76MD77MD80MD81MD82MD123MD124MD125MD126MD127MD128MD167MD179

Other no.

VD372VD383VD185VD375VD374VD183VDt86ELV!ELVi3ELV 16ELV 17ELV19ELV22ELV23ELV25ELV26ELV29ELV34PV41ELVIOELV 11ELV24ELV44ELV12 3ELV 12:7

Host

Capsicum annumLinum usitaliasimumLinum usiiatl.'i.simumLinum u.sitalissimumLinum usitatissimumLinum usitalissimumCapiscum annuumBrassica napus napusHumulus lupulusGossypium hirsutumBrassica napu.s napusBrassica napus napusPiper nigrumSoianum melongeaTrifoUum repensGossypium hirsutumSoianum tuberosumMatricaria chamomiUaBrassica napus napusBrassica napus napusBrassica napus napusBrassica napus napusMatricaria chamomiUaBrassica napus napusBrassica napus napusBrassica napus napusBrassica napus napusMatricaria chamomiUaBrassica napus napusBrassica napus napusGossypium hirsutumGossypium hirsutumCapiscum annumCapiscum annum

Geographic region*

Burgenland/AustriaRostock/GermanyRostock/GermanyRostock/GermanyRostock/GermanyRostock/GermanyBurgenland/Austria

The Netherlands

Sweden

Malchow/GermanyCordoba/SpainRostock/GermanyRostock/GermanyRosenhagen/GermanyKappeln GermanySchonberg, GermanyRostock/GermanyRostock/GermanyEriedrichsfeld/GermanySchonberg/GermanyRostock/GermanyRostock •GermanyRostock GermanyBondelsdorf/GermanyFehmam.̂ GermanyCordoba/SpainCordoba/SpainStockerau/AustriaStockerau/Austria

"No information means that the origin is not known.

mation obtained from agarose gel electrophoresis wasdigitalized by hand to a two-discrete-character-matrix(0 and 1 for absence and presence of RAPD-markers).Phenograms were calculated by using the Jukes-Cantoroption in DNADIST program and application of theFITCH program (Fitch and Margoliash, 1967) to thecomputed distance matrix (PHYLIP package; Fels-enstein, 1989). For running DNADIST, the two discretecharacters 0 and 1 had to be converted to Guanine andThymine in the RAPD data matrix.

Sequencing of the 18SrRNA-Gene

RNAse treated DNA of the haploid isolate MD15 wasamplified using the primers NSO and ITS2 (White etal., 1990). Thirty-one cycles of the program 98 C/15 s;58"C/60 s and 72X/120 s were performed.

For removing the amplification primers, PCR productswere diluted by 3 volumes of TE-buffer and precipitatedovernight by 4 volumes of PEG-buffer (13% w/v poly-ethylene glycol 6000; 1.6 M NaCl). After centrifugation,the pellets were washed in 70% ethanol, dried and dis-solved in 20 /il water. DNA concentrations were esti-mated by agarose gel electrophoresis.

The sequencing reactions were performed by CodonGenetic Systems (Vienna, Austria) using a 373A auto-matic DNA sequencer (Applied Biosystems, Foster City,

CA, USA). Both strands of the 18SrRNA gene weresequenced, using a total of 14 primers, including theprimers NSO and ITS2 engaged in DNA amplification.Up to 400 bases were recorded per sequencing reaction.Sequence primers were optimized by the OLIGO PrimerAnalysis Software (National Biosciences, MA, USA).The sequence of the 18SrRNA-gene from Vertieilliumdahliae is accessible from GenBank under the accessionnumber U33637.

Sequence analyas

The DNA sequence obtained and selected referencesequences derived from GenBank were aligned using theClustalW program (Thompson et al., 1994). Because ofslightly different gene lengths, 'overhanging' nucleotidesat the 3' end had to be cut off for some species. Phylo-genetic computation of ahgnments was done using theprograms DNADIST (Parameter 'Kimura 2'; Kimura,1980), FITCH, SEQBOOT and CONSENSE in thePHYLIP package. The sequence of Saccharomyces cer-evisiae was used as the outgroup. Bootstrap confidencevalues were calculated from 1000 repeats.

ResultsCluster analysis of RAPDs

For RAPD-analysis crude preparations of total nucleicacids were obtained without removing RNA from the

350 el al.

-- 5,080-- 2,840-- 1,700--1.159

-- 0,805

--0.516

KB

1 2 3 4 5 6 7 8 9 10 11 b) 12 13 14 15 16 17 18 19 20 21 22 23 24Fig. 1 RAPD patterns of Verticillium strains. Lane I. MD73: lane 2, MD74; laae 3. MD75: lane 4, MD76: lane 5. MDSI: lane 5. MDS2; lane7. MD80: lane S. MD123; lane 9. MD125; iane 10, .MD126: lane 1 {. lambda DNA digested with Fs/1: lane 12, MD15: lane i i . .MD49: lane 14,MD53: lane 15, MD57; lane 16. MD56: lane 17. MD65: lane IS, MD69: lane 19, MDr27: iane 20, MD128: lane 21. MD63: lane 22, MD55: lane23. MD7i: lane 24. lambda DNA digested with Fsll. (a) Strains isolated from Brassifa napm. primed wilh oligonucleotidc Ml,3. Nole thedistinctive pattern of the strains MD73. MD80 and MD123. (b) Strains from Brassiai napus (MD5^. 57. 561 compared with strains from otherhost plants. Primed with oligonudeotide V5, MD65 (lane 17) is l'\ U'nerum, MD6.1 (iane 20) is V. k'canii. bolh isolated from G. hirsuiwn. Thesetwo strains were not included in the construction of the dendrosram

DNA. PCR-amplifications of DNA from all listed iso-lates were done using the four primers mentioned in theMaterials and Methods section. After separation on a1.3% (w/v) agarose-gel. fragments were obtained for eachisolate and primer, gi%'ing a total of 79 different bands.Examples of the patterns seen are given in Fig. I. Forfurther resolution, a matrix of the 34 strains by absence(0) or presence (1) of RAPD-markers at a given positionwas created by hand (Fig, 2) and analysed wtth the com-puter programs DNADIST and FITCH for estimationof the branch lengths [set to Jukes and Cantor (1969)distance methods within DN.ADIST, and global re-arrangements within FITCH], The resulting phenogramis shown in Fig, 3,

Clusters of P'erticillium isolates showed no strong cor-relation to geographic origin. For example some of theisolates from Rostock, Germany, cltistered together withisolates either from Burgenland. Austria (MD 44,46, 47,48~MD ] 5, 49), others with strains from Cordoba. Spain(MD 70-MD 127, 128), or from Malchow, Germany(MD 77-MD 68).

On the other hand, a distinct, host-specific cluster wasobtained for pathogens of the oilseed rape Brassica napustiapus. independently of local origin. Out of 13 examinedstrains, 10 fell into a clearly distinguished group, rep-resenting isolates from different parts in Germany andSweden. The remaining three .8rfl.s'.v(Ci2-isolates (MD 73,80 and 123) showed no clear group-specific clustering.

Phylogenetjc analysis

For determining the position of V. dahliae among thephylogenetic system of the ascomycetes, sequencing ofthe whole 18SrRNA-gene was chosen because this rep-resents the most conserved ribosomal gene and thereforeit is the most convenient for assessing 'large scale' evol-utionary distances.

For alignment purposes. !8SrRNA-gene sequencesfrom 18 filamentous ascomyceles and deuteromycetesand 2 yeasts (Saccharonnces cerevisiae and Kluy-veromvces laclis) were obtained from GenBank (listed inTab!e'2),

Comparison of the sequences using PHYLIP-com-puter-package leads to a phylogenetic tree shown in F'ig.4, V. dahliae clusters in a clade with the order Hvpocreates(Hvpomvces chrv.Kospermus and Hypocrea hitea)., Mic-roascales (Microascus cirro.su.s and Pseudaltescheria bov-dii) and Phyltachorales {Colletotrichum gloeo.sporiodes.Teleomorph: Glomerella cingukita). Although theinternal resolution of the group would need further analy-sis, this clade is well supported by statistical analysis:bootstrap confidence calculated with 1000 cycles was99.6% for this grouping, giving a good separation fromthe closest clade of the Sordariales., containing Podospora,Sordaria, Neurospora and Chaetomium.

The Ophiosiomatales (O. .'itenocera.s and O. ubrti) clus-ter together with the Diaponhales (Endothia gyrosa andCrvphonectria cuhensis), building up a ciade, which is weliseparated from the former mentioned sister group by97.6% bootstrap confidence.

The grouping of the Dothideates (Septoria nodorum.Aiternaria alternata, Aureohasidium puUulans) and theEiiroliales (Eupenicillium javanicum and Penicillium nol-atum) as sister taxa in the phenogram does not necessarilyreflect a close phylogenetic affinity between these twoclades since the short branch leading to this grotip isnot sufficiently supported. However, the clear distinctionbetween this group which includes taxa without knownteleomorphs {A. puUulans) and the clade which in-cludes Verticiliium clearly demonstrates the need forintegration of the deuteromycetes into the ascomycetoussystem.

The monophyly of the studied filamentous species was

Molecular Characterization of the Plant Pathogen Verticillium 351

Strain Primer M13 Primer GACA

MD73 0101(X)10001I1101000 lKKKXWOOOOllOOlOOOlOOOMD74 1000011100100100000 OOOIOOIOOOOIOOOIIOUOOOMD75 1000011100100100000 OOOIOOIOOOOIOOOIIOUOOOMD76 0000000100100100100 OOOIOOIOOOOIOOOIIOUOOOMD81 1000011100100100000 OOOIOOIOOOOIOOOIIOUOOOMD82 1000011100100100000 OOOIOOIOOOOIOOOIIOUOOOMD80 0101100010101101000 11000000000110010001000M D I 2 3 0100100010011101000 11000000000000010001000MD125 1000011100100100000 OOOIOOIOOOOIOOOIIOUOOOMD126 1000011100100100000 00000010000100011011000MD53 1000011000100000000 OOOIOOIOOOOIOOOIIOUOOOMD57 1000011000100000000 OOOIOOIOOOOIOOOIIOUOOOMD56 lOOOOlIOOOlOOOOOOOO OOOIOOIOOOOIOOOIIOUOOOMD69 0000001000 U U O 1 0 0 0 UOOOOIOOOOUOOIOOOIOOOMD127 0001100101001001000 OUOIOIOIOOOUOOOIOOOOOMD128 0001100101001001000 OUOIOIOIOOOUOOOIOOOOOMD55 01010010001 lUOlOOO UOOOOOOOOO110000001000M D 7 I OIOIOOIOOOUOIOIOOO UOOOOOOOOOOOOIOOOOIOOOM D 7 7 OIOIOOIOOOUOOOOOOO llOOOOOOOOOOOlOlOOOlOOOM D 1 2 4 lOOOOlIOOOlOOOOOOOO OOOIOOIOOOOIOOOIIOUOOOM D 6 8 OIOIOOIOOOUOOOOOOO UOOOOOOOOOOOOIOOOOIOOOM D 7 0 0001100001001001000 OOUOOOOIOOOUIOOOOIOOOM D 5 4 0101001000000000000 1 lOOOOOOOOOOOOUOOOlOOOM D 5 8 0101001000000000001 UOOOOOOOOOOIOUOOOIOOOM D 5 9 0101001000000000001 UOOOOOOOOOUOUOOOIOOOM D 4 4 OIOIOOIOOOUUOOOOO UOOOOOOOOOOOOUOOOIOOOM D 4 5 OlOUOUOOUOOOOOOO UOOOOOOOOOOIOUOOOIOOOM D 4 6 OIOIOOIOOOUUOOOOO UOOOOOOOOOOOOUOOOIOOOM D 4 7 0101001000111100000 UOOOOOOOOOOOOUOOOIOOOM D 4 8 OIOIOOIOOOIUIOOOOO UOOOOOOOOOOOOUOOOIOOOM D 4 9 OIOIOOIOOOUUOOOOO UOOOOOOOOOOOOUOOOIOOOM D 1 5 0101001000U11(X)000 UOOOOOOOOOOOOUOOOIOOOM D 1 6 7 OIOIOOIOOUUIOOOOO UOOOOOOOOOOOOUOOOIOOOM D 1 7 9 OIOIOOIOOUUIOOOOO UOOOOOOOOOOOOUOOOIOOO

Primer VI

loioooioiooooiooOOIOOOOOUOOOOIOOOIOOOOOUOOOOIOOOOOOOOOIOOOOUOOOIOOOOOUOOOOIO00100000110000001010001010000100101000101001010000100000110000000010000011000000OOIOOOOOUOWOOOOOIWXMWUOOOOOO00100000110000000010100010000100OlOOlOOOlOOlOOOO0100100010010000lOlOOOlU 100010010100010100001001010001010000100001000001100000010100010100001010110001010010100101000101001010010100010100101001010001010000100101000101000010010100010100001001010001010000100101000101000010010100010100001001010001010000100

loioooioiooooioo1010001010000100loioooioiooooioo

Primer V5

OlllOlUlOOOOUOOOOOOUUOIUOOOOOIOOOIOOOUUOIUOOOOOIOOOIOOOluioiuoooooioooiooo111 lOlUOOOOO 10001000OOOOOIUOOOOOIOOOIOOOOlllOlUlOOOOUOOOOOOOlllOUUOOOOUOlOOOOUUOIUOOOOOIOOOIOOOOIUOIUOOOOOIOOOOOOOUUOUOOOOOOIOOOIOOOU l 101100000010001000U l 101100000010001000001000111001001000000011111001001000000010o u 111001001000000010OOllOUUOOOOllOOOOOOllUOUUOOOOllOOOOOO011001111001000000000111001UOOOOOOOOOOOOOOUOOl 11100100000000001 lOUOO 1001000000010loioooiuooiooiooooooloioooiuooiooioooooo0010011UOO1000000000OlllOlUlOOOOUOOOOOOOIUOUUOOOIUOOOOOOOIUOUUOOOOUOOOOOOO l U O U l 1000010000000OIUOIUIOOOO10000000OUIOUUOOOOIOOOOOOOOIUOUUOOOOIOOOOOOOOHIO 1111000010000000OIUOUUOOOOIOOOOOOO

Fig, 2 6111817 presence (1)—absence (0) data matrix for RAPD fragments. The matrix ^own is a subset of a work which also included mm-V.dahiiae-str&ins and therefore includes positions with unitorm information

352 MESSNER et al.

napus

\itD1Zi if.chximam.iUa

'i«)5S B.napus

MD81 B.-napus

UD7S B napus

UD74 £.napusUDIZS B.Ttapus

— MD82 B.Tiapus

i£D1Z6 B.napxisMD77 U.cha.m<ymma

MD68 T.mperis

I—ilD71 U.chaTTumMta.

XUD73 B.ntzpus

'MDSS C.hirsutum

<

MDI73 Connuitm

UD167 C.annu-um

UD15 C. annuum

UD44

MD46

itDBB C.hirsutum

.MD70 S.U

^ ,ilBIZ8

MDi 27

\MD12B Chirsrutum

G- n'VT's^ixiTTi

MD1Z3 S.napTis

~MD80 B.napus

il/n.'

MDSS S.TTielongena

UD58 P.nignan.20% Dissimilaiit)'

Fig. 3 Dendrogram of 34 isolates of V. dahliae Kleb. from differenthost plants based on data from RAPD analysis. The dendrogram wascalculated using DNADIST (Jukes-Cantor option) and FiTCH pro-gram of the PHYLIP package. Species names of host plants are indi-cated

corroborated by 100% bootstrap confidence in respectto the unicellular yeasts S. cerevisiae and K. laciis.

DiscussionThirty-four isolates of Verticillium dahliae from nine gen-era of dicotyledonous host plants were studied usingRAPD-PCR. Approximately 90% of the V. dahliae iso-lates fell into two groups (see Fig. 1). One group consistsalmost entirely of V. dahliae isolates from Brassica napus(10 strains) but also includes one isolate from Matricariachamomilla. The isolates were collected in Gennany andSweden. The second group comprises isolates fromdifferent host plants such as Capiscum, Linum, Solanummelongena, S. tuberosum, Trifolium, Humulus, Piper, andGossypium coliected from different european regions. The

second group also contains three isolates from Brassicanapus. Rohde (1995) recently tested these Brassica napusisolates for virulence. Isolates MD 73 and MD 80 showeda significantly lower aggressiveness than did the otherisolates from B. napus when inoculated into seedlings ofB. napus grown on quartz-sand. Similar data werereported from Horiuchi et al. (1990). Their isolates ofgroup D were only pathogenic to brassicaceous hostplants. Jackson and Heale (1985) demonstrated naturalstable diploids of V. dahliae from Brassica spp. Measure-ments of the conidium length by Rohde (1995) indicatethat conidiospores of all the Brassica isolates except MD80 may belong to the var. longisporum which was con-sidered to be a diploid race of V. dahliae by Jackson andHeaJe (1985). In V. albo-atrum, host specificity appearsto be the basis of a differentiation in two groups. Okoliet al. (1993) included in group 1 all the isolates fromlucerne (alfa-alfa), whereas group NL ( = non-lucerne)consists of the isolates of severai other host plants.

According to Domsch et al. (1980) and Gams andvan Zaayen (1982) the anamorphic genus Verticilliumis heterogenous. The type species Verticillium tenerum(Nees) Link possesses a teleomorph in Nectria invemaPethy. (Hypocreales). Cordyceps and Torrubiella are twoclavicipitalien genera with a Verticillium anamorph. ln afirst phylogenetic approach we have been able to growV. dahliae from Brassica and Capsicum in a unicellularyeast-like growth stage using a Czapek-Dox medium.Purified cell walls of this unicellular growth stage showedthe Glucose Mannose Galactose pattern (Glc: 72%, Man:10%, Gal: 18%) and a ubiquinone Q-10 H2. A similar cellwall composition and ubiquinone spectrum was found inPringsheimia chamaecyparidis ( G l c ; 7 6 , M a n : I I , G a i :13), a dothidealean fungus (unpublished data). To clarifyif the carbohydrate pattern of yeast stages from fila-mentous Ascomycetes are phylogenetically useful, wehave sequenced the whole ]8SrDNA of V. dahliae andcreated a phylogenetic tree (Fig. 4). V dahliae clusters ina clade with the order Hypocreales.

This ciose relation to the Hypocreales was further cor-roborated with the finding, that a 1044 bp-fragment ofthe ! 8SrRNA-gene from Hypocrea schweinitzii showedthe highest score in a blast-search of the V. dahliae-genethrough the GenBank. Because of non-completeness ofthe sequence, H. schweinitzii was not included in theconstruction of the phylogenetic tree.

No affinities of V. dahliae to the Dothideales werefound. Although yeast cell wall sugars and the ubiqui-none system are very usefui characters in the taxonomyof yeasts, their significance is probably rather low in thetaxonomy of filamentous Ascomycetes.

Acknowledgements

For sending VerticilUum strains we have to thank Prof. E Schlosser,Dr. B. Fortnagl and Dr. D. Amelung. We also express out sinceregratitude to K. Lopandic for the analysis of the cell wall sugars. Thiswork was supported by the FWF-grant P09255-BIO, the 'EU-Vorbereitungsprojekf from the Bundesministerium fOr Wissenschaft,Forschung und Kunst, Austria, and the 'Jubilaumsfonds derOsterreichischen Nationalbank', Austria.

Molecular Characterization of the Plant Pathogen Vertieillium 353

Table 2Sources of 18SrRNA-gene sequence data

SpeciesGenBankaccession no. Reference

Ophiostoma stenocerasOphiostoma ulmiEndolhia yyrosaCryphonectria cubensisHypocrea luteaHypomyces chrysospermusVertieillium dahliaePseudalle.'icheria boydiiMicroascus cirrosusColletotrichum gheosporiodesPodospora anserinaSordaria fimicolaNeurospora crassaChaetomium elatumSeptoria nodorumAlternaria alternataAureohasidium pullulansEupencillium JavanicumPenciWum notatumSaccharomyces cerevisiaeKluYveromvces lactis

M85054M8326IL42443L42439D14407M89993U33637M89782M89994M55640X54864X69851X04971M83257U04236U05194M55639U21298M55628JO1353X5183O

Berbee and Taylor (1992c)Berbee and Taylor {1992c)Chen et al., unpublishedChen el al., unpublishedYoshimura and Sugiyama, unpublishedBerbee and Taylor (1992b)This paperBerbee and Taylor (1992b)Berbee and Taylor (1992b)lllingworthet al. (1991)Sogin, unpublishedWilmotteetal.(l993)Sogin etal. (1986)Berbee and Taylor (1992a)Morales et al., unpublishedJasalavich et al.. unpublishedIllingworth etal. (1991)Berbee et al., unpublishedSogin et aL. unpublishedMankin etai. (1986)Maieszka and Clark-Walker (1990)

Ophiostotn.a,

O-phio.ulmi

t Endothiaioo

CryphoTiectria.

Hyp OTnyces

•V.daMiae

- MicToascits

- CQlLetotrichxtTn

PodospoTa

53.9 '^Ch,aetoTnixi7n

Septoria

-Alternaria

E^eniciHvwm

PenidtiiiLmrftt

- SaccharoTnyces

— Kltiyveromyces

Fig. 4 An estimation of the pbylogenetic tree of 21 fungi and yeasis(see Table 2) derived from complete 18SrRDNA sequences. The algo-rithms used for tree construction are referred to in Materials andMethods. Bootstrapping values were calculated from 1000 cycles

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