SUMMARY

Random Amplified Polymorphic DNA (RAPD)analysis and mating type distribution were used to in-vestigate the population biology of the phytopathogenicfungus Uncinula necator (Schw.) Burr., the causal agentof powdery mildew of grapevine. It is known that thefungus has two overwintering strategies, mycelium andconidia in dormant buds or cleistothecia, but their rela-tive importance in disease epidemiology is still undeter-mined. Recently, the existence has been hypothesized oftwo genetically separated biotypes in U. necator thatwould be related with its overwintering modes: a bio-type overwintering as conidia and mycelium in budswould infect shoots and leaves early in the season; theother biotype would overwinter as cleistothecia and in-fect bunches. RAPD analysis was carried out on 374 iso-lates of U. necator collected in Southern Italy. Statisticalanalysis of variation clustered the isolates into two ma-jor groups according to the time in the season whenthey were sampled in vineyards, confirming the exis-tence of two different biotypes into the fungal species.Similar proportions of the two mating types were foundin the pathogen’s populations, as well as into each bio-type, even in single vineyards. Therefore, the two ma-ting types of the fungus are not separated either inspace or time. Sexual crosses between isolates belongingto the two biotypes were fertile and yielded viableascospore progeny. These results suggest that meioticrecombination can be an important source of geneticvariation in U. necator and cleistothecia can play an im-portant role in its overwintering.

Key words: Uncinula necator, grapevine, RAPDanalysis, sexual behaviour, genetic variability, epidemiol-ogy.

INTRODUCTION

Powdery mildew, caused by Uncinula necator (Schw.)Burr. [teleomorph of Oidium tuckeri Berk.; recently the

Corresponding author: F. FaretraFax: +39.080.5443052E-mail: faretra@agr.uniba.it

fungus was renamed Erysiphe necator Schw. and placedinto the section Uncinula of the genus) (Braun andTakamatsu, 2000)], is a worldwide economically impor-tant fungal disease of the grapevine. It costs millionsdollars annually to vine growers, due to crops losses andan intensive usage of fungicides for its control.

Only few genetic studies are available in U. necatordue to its obligate biotrophism. The fungus cannot begrown on artificial media and observations in the fieldcan be carried out only in a limited period of the year.Field- or greenhouse-grown vines and detached leaveshave been used to study different aspects of the dis-ease (Doster and Schnathorst, 1985; Chellemi andMarois, 1991). For molecular investigations, however,these methods are subject to the possible interferenceof other epiphytic micro-organisms. Although more la-borious, the usage of dual culture for growing U. neca-tor colonies on in vitro-grown grape material underaseptic conditions is a more reliable technique(Klempka et al., 1994; Evans et al., 1996; Miazzi et al.,1997).

U. necator has been reported to overwinter as myce-lium or conidia in dormant buds (Sall and Wrysinsky,1982; Pearson and Gärtel, 1985; Gemmrich and Seidel,1996) and/or as cleistothecia on infected tissues, on thebark of vines or in the soil (Diehl and Heintz, 1987;Pearson and Gadoury, 1987; Gadoury and Pearson,1988, 1991; Viccinelli et al., 1996; Cortesi et al., 1997).However, the role of the sexual process in the pathogen’slife cycle and epidemiology of powdery mildew, and itsimportance in the generation of new recombinant geno-types has been neglected for a long time. In Europe,cleistothecia were not observed for 40 years after the in-troduction of the pathogen from America, so that itsoverwintering was believed to be sustained exclusivelyby dormant mycelium and conidia. The sexual system ofthe fungus is a bipolar heterothallism coded by the ma-jor gene MAT1 with its two alleles (likely idiomorphs,based on what known on other Ascomycetes) MAT1-1and MAT1-2 (Gadoury and Pearson, 1991; Miazzi et al.,1997, 2002). The initial introduction of only one matingtype was probably the reason of the delay of cleistotheciadiscovery in Europe (Couderc, 1893; Yarwood, 1957)and, more recently, in Peru (Dongo and Aréstegui, 1973)and Australia (Wicks et al., 1985).

Genetic markers are crucial for studying the genetic

Journal of Plant Pathology (2003), 85 (2), 123-129 Edizioni ETS Pisa, 2003 123

OBSERVATIONS ON THE POPULATION BIOLOGY OF THE GRAPE POWDERY MILDEW FUNGUS UNCINULA NECATOR

M. Miazzi, H. Hajjeh and F. Faretra

Dipartimento di Protezione delle Piante e Microbiologia applicata, Università degli Studi di Bari,Via Amendola 165/A, 70126 Bari, Italy

structure of pathogen populations and the epidemiologyof the incited disease; molecular markers can be partic-ularly useful for biotrophic fungi, such as U. necator(Milgroom, 1997). Information on population geneticsand epidemiology can result in deeper knowledge ofpopulation biology that could be essential for develo-ping more effective and environmental-friendly diseasecontrol strategies (Milgroom, 2001).

Random Amplified Polymorphic DNA (RAPD)analysis (Williams et al., 1990), a fingerprinting tech-nique based on Polymerase Chain Reaction (PCR), hasbeen successfully applied to study variation in manyfungal pathogens, including U. necator (Délye et al.,1995, 1997, 1998; Evans et al., 1996; Stummer et al.,1999, 2000).

Délye et al. (1997, 1998), through RAPD analysis,distinguished two biotypes in European powderymildew populations having different overwinteringmodes. One biotype was constituted by isolates over-wintering as conidia or mycelium in buds and causingthe typical symptoms on young shoots known as “flagshoots”, early in the season. The other biotype wouldoverwinter as cleistothecia and be responsible of late in-fections mostly on bunches. The two biotypes werethought to be genetically separated because in sexualcrosses they did not yield fertile cleistothecia and viableascospores (Délye et al., 1997, 1998).

The aim of the present work was to investigate thepopulation biology of U. necator in some vineyards ofSouthern Italy, using mating type and RAPD markers.Special attention was paid to the pathogen’s sexual be-haviour and the occurrence of distinguishable biotypesin its natural populations.

MATERIALS AND METHODS

U. necator isolates. Thirty-one vineyards were select-ed as representatives of the most important grape-grow-ing areas in six regions of Southern Italy (Abruzzo,Basilicata, Calabria, Molise, Puglia and Sicilia). Samplesof naturally infected bunches, leaves and canes werecollected between April and September from 1998 to2000. Isolates from typical flag shoots were sampled im-mediately after their appearance (April-May). An exten-sive sampling (140 isolates) was carried out in an experi-mental vineyard made up by randomly repeated plots ofeight cultivars, recording the exact position of eachsample.

Plantlets of the very susceptible table grape cv. Bare-sana were grown and dual cultures of U. necator ongrape leaves established as described by Miazzi et al.(1997). Briefly, young expanded leaves were collectedfrom in vitro-grown grapevine shoots and placed singlyon a 2-mm thick layer of HB medium (Hoos and Blaich,1988), with half strength of salts, in 55-mm-diam Petridishes, taking care that their stalks were immersed intothe medium. Leaves were seeded with small amounts of

conidia collected from naturally infected samples withsingle eyelashes glued to glass pipettes. Dishes weresealed with Parafilm® and kept at 21±1°C with a 16 hphotoperiod from a combination of 3 Osram L36Wlamps and 3 Sylvania Grolux F36W lamps. After oneweek, single conidia were collected and transferred ontonew leaves to obtain pure cultures. Isolates were main-tained by transferring conidia from single chains ontonew leaves at 4-5 week intervals (Fig. 1).

Mating type. The strains UN52 (MAT1-1) and UN68(MAT1-2) were used as references to assess the matingtype of newly collected isolates (Miazzi et al., 1997).Each isolate was paired with both reference strains onsingle leaves that were kept under above described con-ditions. Cleistothecia appearance was checked at one-week interval for 6-7 weeks with the aid of a stereomi-croscope. Mature cleistothecia were collected with aneedle, squashed in a drop of a vital stain (1 µl of astock solution containing 2 mg ml-1 fluorescein diac-etate in acetone diluted in 1 ml of water) on a glass slide(Widholm, 1972) and observed with the aid of an epi-fluorescence microscope (Photomicroscope III, Zeiss,equipped with a HBO 100 W/2 mercury lamp; excita-tion filter BP436/8, beam-splitter FT460, barrier filterLP470) to assess ascospore viability.

DNA extraction and RAPD amplifications. Myce-lium and conidia (approximately 4 mg fresh weight)were collected from 2-3-week-old colonies by scrapingthe surface of infected grape leaves with a disposablelancet. DNA was extracted by using InstaGene Matrix(Bio-Rad Laboratories, Hercules, CA, USA), followingthe manufacturer’s instructions. DNA solutions werekept at -20°C until usage. Samples of DNA from twostrains of U. necator, kindly supplied by Prof. Corio-Costet (National Institute of Agricultural Research, Bor-deaux, France), were used as representatives of the two

124 Population biology of Uncinula necator Journal of Plant Pathology (2003), 85 (2), 123-129

Fig. 1. A dual culture of Uncinula necator on a leaf from invitro-grown grape plantlets.

biotypes: F1 belonged to the “flag shoot” biotype andF3 to the “ascospore” biotype (Délye et al., 1998).

RAPD amplifications were carried out in 25 µl con-taining 10 mM Tris-HCl, pH 9.0; 50 mM KCl; 0,1% Tri-ton X-100; 2 mM MgCl2; 75 µM each of dATP, dCTP,dGTP and dTTP (Promega, Madison, WI, USA); 0,5µM primer; 100 ng of target DNA and 2 U of Taq DNAPolymerase (Promega, Madison, WI, USA). Reactionswere carried out in a thermal cycler (Geneamp PCR Sys-tem 9700; Perkin Elmer, Norwalk, USA) programmed asfollows: 4 min at 95°C (initial denaturation); 40 cycles of30 s at 94°C, 30 s at 35°C, 30 s at 72°C; and a conclusiveextension phase of 7 min at 72°C.

Sixty-three 10-mer primers (Kit A, B, D; OperonTechnologies, Alameda, CA, USA) were used for DNAamplification from a first set of 28 isolates. Twenty-three

primers revealing higher polymorphism were used toamplify DNA extracted from the remaining isolates.Two independent amplifications were done for each iso-late and primer combination.

Aliquots (10 µl) of reaction mixtures were loaded on1,4% Amplisize agarose gel (Bio-Rad Laboratories,Hereules, CA, USA) and run in 0,5xTBE buffer (45mM Tris-borate, 1 mM Na-EDTA; pH 8) at 110 V for110 min (Sub-Cell TM, Bio-Rad Laboratories, Hercules,CA, USA). A 100-bp DNA Ladder (New England Bio-Labs, Beverly, USA), giving 12 bands between 100 and1,500 bps, was used as a standard for molecular sizes.Gels were stained with 1 µg ml-1 ethidium bromide for40 min, destained in water for 10 min, and gel imageswere digitalised with a Gel Doc 1000 System (Bio-RadLaboratories, Hercules, CA, USA).

Journal of Plant Pathology (2003), 85 (2), 123-129 Miazzi et al. 125

Table 1. Origin of Uncinula necator isolates.

(a) Alphonse Lavallèe, Baresana, Italia, Negroamaro, Regina bianca, Sangiovese, Trebbiano, Uva di Troia. (b) Malvasia nera, Montonico nero.

VineyardNo.

Location Cultivar No. ofisolates

Isolatecode

Sampling time Source

1 Valenzano (Bari) Several (a) 140 V-isolates July 1998 Bunches2 Locorotondo (Bari) Several (b) 3 304,305,309 August 1999 Bunches

3 Sannicandro (Bari) Regina bianca 3 290,293,294 July 1999 Bunches4 Brindisi (Brindisi) Negroamaro 10 16-25 May 1999 Flag shoots5 Cellino (Brindisi) Malvasia nera 3 47,48,49 June 1999 Flag shoots6 Cellino (Brindisi) Negroamaro 6 51-55,57 June 1999 Flag shoots7 Cellino (Brindisi) Negroamaro 12 58-69 June 1999 Flag shoots8 Oria (Brindisi) Negroamaro 28 UO1-UO28 May-June 2000 Flag shoots

9 Trinitapoli (Foggia) Michele Palieri 7 1-7 May 1999 Flag shoots10 Cannole (Lecce) Negroamaro 2 314,315 September 1999 Bunches11 Galatone (Lecce) Malvasia nera 14 30-36,39-44,46 May 1999 Flag shoots12 Ginosa (Taranto) Italia 7 93-99 July 1999 Leaves13 Vinosa (Taranto) Trebbiano 3 161-163 July 1999 Bunches14 Ginosa (Taranto) Italia 56 G1-G55, GG5 July 1999 Bunches

15 Massafra (Taranto) Victoria 3 251,256,260 July 1999 Bunches16 Castellana (Pescara) Michele Palieri 8 1 77-81,205-207 July 1999 Bunches17 Collalto (Pescara) Montepulciano 8 261-264,274-277 July 1999 Bunches18 S. Martino (Termoli) Montepulciano 4 221-224 July 1999 Bunches19 S. Martino (Termoli) Montepulciano 6 225-230 July 1999 Bunches20 S. Martino (Termoli) Montepulciano 3 239,245,246 July 1999 Bunches

21 Nova Siri (Matera) Pinot bianco 16 107,110,112-125 June 1999 Leaves22 Cirò marina (Cosenza) Gaglioppo 5 76,80,82,85,86 June 1999 Flag shoots23 Cirò marina (Cosenza) Gaglioppo 5 88-92 June 1999 Flag shoots24 Cirò marina (Cosenza) Gaglioppo 4 100-103 June 1999 Leaves25 Cirò marina (Cosenza) Gaglioppo 3 104-106 June 1999 Leaves26 Cirò marina (Cosenza) Gaglioppo 4 126,129,130,132 June 1999 Leaves

27 Cirò marina (Cosenza) Gaglioppo 5 135-139 June 1999 Leaves28 Chiaramonte (Ragusa) Italia 2 150,155 July 1999 Leaves29 Cannizzaro (Ragusa) Italia 1 156 July 1999 Bunches30 Chiaramonte (Ragusa) Italia 1 157 July 1999 Bunches31 Primosole (Catania) Italia 2 159,160 July 1999 Bunches

Data analysis. Manipulation of gel images and elabo-ration of data were carried out using the software pack-age Diversity Database™ (Ver. 2.1 for Windows; Bio-Rad Laboratories, Hercules, CA, USA). The bandingpatterns of each isolate was scored for the presence orabsence of each marker. To provide a quantitative mea-sure of relatedness among isolates, genetic similarity be-tween all pairs of isolates was estimated according to theformula 2bij/(bij + bi + bj), where bij is the number ofamplicons shared by two isolates i and j, and bi and bjare the number of unshared bands (Dice, 1945; Nei andLi, 1979). Similarity matrices were then used to con-struct a phylogenetic tree according to the UnweightedPair Group Method using Arithmetic Averages (UPGA-MA). Data were also submitted to Principal Compo-nents Analysis.

RESULTS AND DISCUSSION

A collection of 374 isolates of U. necator was estab-lished from samples of naturally infected grape tissuescollected in 31 vineyards located in 6 regions of SouthItaly (Abruzzo, Basilicata, Calabria, Molise, Puglia andSicilia).

All the isolates of U. necator were singly mated withtwo reference strains of known mating type, UN52(MAT1-1) and UN68 (MAT1-2). The number of clei-stothecia in fertile crosses was broadly variable depend-ing on isolates and crosses; some crosses yielded nu-merous cleistothecia, scattered all over the fungalcolonies, while others yielded only few cleistothecia. Allisolates proved fertile with only one of the referencestrains. No isolates were able to differentiate cleistothe-cia in crosses with both the reference strains, or whenthey were grown alone on grapevine leaves. These re-sults corroborate previous findings on the sexual be-haviour and mating system of U. necator that can be as-cribed to a bipolar heterothallism under the control ofa single major gene, MAT1, with its two alleles MAT1-1and MAT1-2 (Gadoury and Pearson, 1991; Miazzi etal., 1997, 2002).

The two mating types, MAT1-1 and MAT1-2, werepresent in similar proportions in all pathogen popula-tions, although the former prevailed slightly over thelatter: 199 isolates were MAT1-1 and 175 were MAT1-2 (Table 2). The two mating types were present insimilar proportions even in single vineyards (Table 2).Numerous isolates (140) were sampled in an experi-mental vineyard made up by vines of 8 cultivars. Mat-ing types were evenly present on all the cultivars andshowed an aggregated distribution in the vineyard(Fig. 2).

In preliminary RAPD experiments carried out with28 fungal isolates, 23 out of 63 tested primers generat-ed from 1 to 10 amplicons that separated in agarose gelinto discrete bands corresponding to molecular sizesranging from 200 to 1,300 bps (Fig. 3). RAPD profiles

126 Population biology of Uncinula necator Journal of Plant Pathology (2003), 85 (2), 123-129

Table 2. Distribution of mating types in Uncinula necator iso-lates of different origin.

No. of isolatesVineyard No. No. of testedisolates MAT1-1 MAT1-2

1 140 80 602 3 3 03 3 0 34 10 4 65 3 1 26 6 3 37 12 3 98 28 16 129 7 5 210 2 2 011 14 4 1012 7 3 413 3 1 214 56 24 3215 3 0 316 8 3 517 8 6 218 4 2 219 6 4 220 3 3 021 16 6 1022 5 3 223 5 4 124 4 4 025 3 2 126 4 3 127 5 4 128 2 2 029 1 1 030 1 1 031 2 2 0

Total 374 199 175

Fig. 2. Distribution of mating types, MAT1-1 (dark grey) andMAT1-2 (light grey), in the population of Uncinula necator inan experimental vineyard made up by 8 grape cultivars. Eachcell represents a vine: I = ‘Italia’; S = ‘Sangiovese’; L =‘Alphonse Lavallèe’; R = ‘Regina’; N = ‘Negroamaro’; U =‘Uva di Troia’; B = ‘Baresana’; T = ‘Trebbiano’.

were unchanged even when PCR reaction was carriedout on DNA extracted from the same isolates one yearlater, i.e. about after 12 asexual generations by colonytransfer. Contamination by grapevine DNA was neverdetected in RAPD amplifications (data not shown).

The 23 selected primers were used with the wholecollection of 374 isolates of U. necator. These yielded137 amplicons, 55 of which were polymorphic. Poly-morphic markers were used to establish genetic relat-edness among fungal isolates. RAPD analysis showed alimited genetic variation in U. necator, the mean valueof genetic similarity in the whole population being0.87.

The UPGMA cluster analysis discriminated two ma-jor groups of isolates (Fig. 4). Group I included all the53 isolates collected from “flag shoots” and 30 isolatessampled from leaves or bunches early in the season,from May to the beginning of June; intra-group meangenetic similarity was 0.90. Group II included the re-maining 291 isolates collected later in the season; intra-group mean genetic similarity was 0.84. Inter-group ge-netic similarity was 0.82, only slightly lower than that ofthe whole population. Usually, into each of the two ma-jor groups, isolates sampled in a same vineyard clusteredtogether (Fig. 4). Two French reference strains of thebiotype “flag shoot” and “ascospore” (Délye et al., 1997)clustered in Group I and II, respectively. The existenceof the two distinguishable groups of isolates was alsoconfirmed by Principal Components Analysis (Fig. 5).

Journal of Plant Pathology (2003), 85 (2), 123-129 Miazzi et al. 127

Fig. 3. Examples of banding patterns obtained using two sin-gle 10-mer primers (OPD5, left; OPD8, right) to amplifyDNA from in vitro-grown isolates of Uncinula necator. Ar-rows indicate markers discriminating isolates belonging to thetwo hypothesized biotypes of the pathogen.

Fig. 4. Phylogenetic tree derived by UPGMA analysis ofRAPD data: Group I: isolates derived from flag shoots (A in-dicates isolates from flag shoots from a single vineyard).Group II: isolates derived from leaves and bunches after may;letters B to F indicate sub-groups of the Group II, formed inthe experimental vineyard N. 1. Letter G indicates isolatesfrom the vineyard N. 14.

Fig. 5. Principal component analysis of variation in U. necatorisolates on the ground of RAPD markers. Notice the discrimi-nation of isolates from flag shoots (F) from those collectedlater in the season and likely deriving from ascospores (A).

A

F

These results corroborate previous findings on the exis-tence of two distinct biotypes in U. necator (Délye et al.,1997, 1998).

Isolates belonging to Group I were found only untilJune. This suggests that such variant of the pathogen,which is prevalent early in the season, tends to disap-pear, showing a very low, if any, frequency into the fun-gal population later in the season. To explain the phe-nomenon, Délye et al. (1997) hypothesized that the ac-tive stage of the “flag shoot” biotype is limited tospring, when it infects primarily shoots and leaves; thenit would survive in latent form into infected buds untilthe next spring, when new infections occur.

Mating types were similarly distributed into the twomajor groups of isolates: 37 and 46 isolates were, respec-tively, MAT1-1 and MAT1-2 in Group I (“flag shoot”biotype); 162 and 129 isolates were, respectively, MAT1-1 and MAT1-2 in Group II (“ascospore” biotype).

Délye et al. (1998) crossed isolates belonging to thetwo different biotypes and observed differentiation ofcleistothecia but not of viable ascospores; hence, theyconcluded that the two biotypes of the fungus are genet-ically isolated since gene recombination allowed by thesexual process is prevented. On the contrary, in ourstudy, crosses between isolates of opposite mating typesrecognized as belonging to the “flag shoot” and the“ascospore” biotype were fertile, forming normal cleis-tothecia and ascospores within 7 weeks. Ascosporesproved normally viable when stained with the vital stainfluorescein diacetate. Cortesi et al. (personal communi-cation) obtained similar results in crosses among U.necator isolates from Tuscany. Therefore, genetic separa-tion of the two biotypes is not due to sexual sterility, andthe sexual process can occur in nature. Likely, the differ-ent ecological niches they occupy result in a temporalseparation preventing mating. The “flag shoot” biotype,active only early in the season, surviving in infected budsand in a quiescent state in late summer, might be im-paired in recombining with the “ascospore” biotype thatis active later in the season when the sexual process oc-curs and cleistothecia are differentiated in vineyards.

Further observations on this aspect are in progress inthe hope that more environment- and consumer-friend-ly crop protection strategies can be designed with adeeper knowledge on U. necator population biology.For instance, spraying early in the season could be use-less if the “flag shoot” biotype is not really responsibleof infection of bunches. Biotype-specific markers havebeen used for developing SCAR (Sequence Charac-terised Amplified Regions) primers for epidemiologicalstudies (Délye et al., 1997; Hajjeh et al., unpublished re-sults). These should allow following the evolution of thecomposition of U. necator populations during thegrapevine-growing season and to understand the rolesof the two biotypes in the pathogen’s life cycle and inthe epidemiology of grape powdery mildew.

The combination of mating types and RAPD markersproved effective in the study of the genetic structure of

the pathogen’s population present in an experimentalvineyard with 8 cultivars. In this experimental vineyard,RAPD analysis revealed the existence of 5 groups of iso-lates (B-F) characterised by intra-group genetic similari-ty higher than 0.94; they showed a clear aggregation inthe vineyard, resembling that of mating types (data notshown). Mating types were distributed in the 5 groupsas follows: 6 and 23; 23 and 1; 12 and 16; 10 and 19; 24and 6; respectively for MAT1-1 and MAT1-2.

Both types of markers showed that similar isolatesshowed an aggregated distribution in the vineyard, in-dependently from cultivars. This result supports the hy-pothesis formulated by Bulit and Lafon (1978) andWilloquet (1994), according to which, whatever theprevalent overwintering mode, the spreading of U. neca-tor clones in vineyards would be rather limited and atshort distance, in contrast with the nature of its wind-dispersed conidia. The dissemination of the fungus overlong distances would be more likely due to man’s activi-ties (Délye et al., 1997).

The similar distribution of mating types observed inthe pathogen’s populations in Southern Italy indicatesthat there are no obstacles, neither geographical nortemporal, that prevent matching of sexually compatibleindividuals. Hence, the occurrence of the sexualprocess, leading to differentiation of cleistothecia andascospores, depends exclusively on the occurrence ofpermissive environmental conditions, and may play animportant role in the population biology of U. necator,acting as a source of release of new recombinant geno-types and increasing the inoculum responsible of pri-mary infections in the next season.

ACKNOWLEDGEMENTS

Research granted by the Minister of Agricultural andForestry Politics, project “Genetic variability of fungalpathogens and selection for resistance of grapevine tobiotic stress” (Coordinated Research “Assisted selectionfor resistance to biotic stress and qualitative improve-ment of fruit trees”, National Programme on PlantBiotechnology), by the University of Bari, project “Epi-demiology and genetics of phytopathogenic microor-ganisms”, and by CEGBA (Centro di Eccellenza in Ge-nomica Comparata in Campo Biomedico e Agrario), Re-search Line n. 7. We thank Prof. M.F. Corio-Costet(National Institute of Agricultural Research, Bordeaux,France) who kindly provided us with DNA of represen-tative strains of the two biotypes of U. necator.

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Received 3 March 2003Accepted 9 June 2003