Structure of Melampsora larici-populina populations on wild and cultivated poplar

Populations of Melampsora larici-populina, causal agent of poplar leaf rust, have been collected during 1992–1994 in three types of sites: natural stands of Populus nigra in the south-east of France; cultivated poplar stands in the northern half of France, including mainly interspecific and exotic hybrids of poplar; and two overseas populations collected from Northern Ireland and Western USA. The pathotype of each rust isolate was determined by inoculating a differential set of poplar clones. The number of virulences per isolate and the frequency of each virulence in each population were compared, according to the sites and to the clones from which the isolates were collected.Several diversity indices were applied to the populations. The and Simpson indices were retained since they described best the richness and the evenness, respectively, of the populations, and they were the least sensitive to sample size. The complexity of races was lower in the natural stands of P. nigra than in the cultivated poplar stands. Populations of M. larici-populina collected on Populus × euramericana Robusta and P. nigra italica (Lombardy poplar) presented a high richness and evenness, confirming the value of these clones for describing race populations. Populations from Western USA presented a very low diversity, which is in accordance with the recent introduction of the pathogen in North America. These results suggest that the race populations of M. larici-populina are mainly influenced by the structure of the host populations.     

Genetic variability of Colletotrichum lindemuthianum in wild populations of common bean

The genetic structure of wild populations of Colletotrichum lindemuthianum was evaluated for virulence and molecular markers. Forty-five isolates were collected from five wild common bean populations located in their South-Andean centre of origin. The five pathogen populations were monomorphic in their ITS regions, but 45 polymorphic markers were identified using RAPDs. Polymorphism for virulence was also observed; 15 pathotypes were characterized on an international set of 12 differentials. A molecular variance analysis ( AMOVA ) showed that a very large part of the total genetic variation was within populations. Statistical analysis showed that there was a weak though significant differentiation among the five populations for the RAPD and virulence markers. A positive and significant correlation was found between geographic distance and the distances from RAPD and virulence data, suggesting migration between adjacent populations along the Argentinian transect. Our results suggest that the Andean wild isolates of C. lindemuthianum do not reflect all the putative diversity found in the isolates from cultivated common bean.     

Genetic Diversity and Pathogenic Variation of Colletotrichum lindemuthianum in the Three Centers of Diversity of Its Host, Phaseolus vulgaris

ABSTRACT Population subdivision of Colletotrichum lindemuthianum, the causal agent of anthracnose, was studied in three regions located in three centers of diversity of its host, Phaseolus vulgaris. Random amplified polymorphic DNA (RAPD) markers, restriction endonuclease analysis of the amplified ribosomal internal transcribed spacer region, and virulence on a set of 12 cultivars were used to assess the genetic diversity of C. lindemuthianum strains isolated in Mexican, Ecuadorian, and Argentinean wild common bean populations. The three regions were significantly differentiated for molecular markers. For these markers, Mexico was the most polymorphic and the most distant from Ecuador and Argentina. The majority of the RAPD alleles present in Ecuador and Argentina were found in Mexico, suggesting that Andean populations have been derived from the Mesoamerican center. Pathogenicity tests on a set of 12 cultivars showed that all but one of the Mexican strains were virulent exclusively on Mesoamerican cultivars. Argentinean strains were virulent preferentially on southern Andes cultivars, and the Ecuadorian strains, except for one strain, were avirulent on all cultivars. These results suggest an adaptation of strains on cultivars of the same geographic origin. Thus, based on molecular and virulence markers, C. lindemuthianum strains isolated from wild common bean populations were divided into three groups corresponding to host gene pools.     

Genetic Structure of Mycosphaerella populorum (Anamorph Septoria musiva) Populations in North-Central and Northeastern North America

ABSTRACT In order to characterize the genetic variation of the poplar pathogen Mycosphaerella populorum (anamorph Septoria musiva), we have studied seven North American populations using the polymerase chain reaction random amplified polymorphic DNA (RAPD) technique. The fungal populations were sampled in 2001 and 2002 by obtaining 352 isolates from cankers and leaf spots in hybrid poplar plantations and adjacent eastern cottonwood (Populus deltoides). A total of 21 polymorphic RAPD markers were obtained with the six RAPD primers used. A fine-level scale analysis of the genetic structure within the populations revealed that subpopulations sampled on P. deltoides and on hybrid trees were not significantly differentiated. In contrast, analyses performed on the entire data set showed high levels of haplotypic diversity and moderate to high genetic differentiation, with 20% of the expected genetic diversity found at the interpopulation level. Moreover, a high and significant correlation between genetic and geographic distances among populations was found, suggesting isolation by distance of the sampled populations. Although the occurrence of the sexual stage of this fungus remained unclear in field populations, five of the six populations were at gametic equilibrium for RAPD loci, suggesting the occurrence of recombination episodes in Septoria musiva populations. Overall, S. musiva appears to consist of differentiated subpopulations, with both asexual and sexual recombination contributing to the local level of genetic structure.     

Genetic Diversity in Relation to Sexual and Asexual Reproduction in Populations of Melampsora larici-epitea

Genetic diversity of Melampsora larici-epitea leaf rust from three cultivated stands of the willow Salix viminalis was studied using AFLP polymorphisms at 60 loci. One population was located in Northern Ireland and two in Sweden. Analysis of molecular variance (AMOVA) showed that most of the genetic variation was distributed on a fine scale within the field in all populations. Both Swedish populations displayed a very high genotypic diversity (normalized Shannon's indices of 0.95 and 1.00) and random association among loci. These results suggested that sexual reproduction had an important influence on the Swedish populations. The occurence of the alternate host (larch) adjacent to one of the Swedish rust populations did not affect the genetic diversity. However, severe rust attacks started earlier in the season in this population. The M. larici-epitea population in Northern Ireland was characterized by a low genotypic diversity (normalized Shannon's index = 0.54) and non-random association among loci was indicated by test of multilocus association and by pairwise tests among loci. These results suggested that asexual reproduction had a major effect on the genetic structure of this population, probably because of the overwintering of asexual spores and/or a population bottleneck associated with the annual sexual phase.     

Genetic Diversity in Colletotrichum gloeosporioides from Stylosanthes spp. at Centers of Origin and Utilization

ABSTRACT Using molecular markers, this work compares the genetic diversity in Colletotrichum gloeosporioides infecting species of the tropical forage legume Stylosanthes at the center of origin in Brazil and Colombia with that of Australia, China, and India, where Stylosanthes spp. have been introduced for commercial use. There was extensive diversity in the pathogen population from Brazil, Colombia, China, and India. The Australian pathogen population was least diverse probably due to its geographical isolation and effective quarantine. The extensive diversity in China and India means that threats from exotic pathogen races to Stylosanthes pastures can potentially come from countries outside the South American center of origin. In Brazil and India, both with native Stylosanthes populations, a high level of genetic differentiation in the pathogen population was associated with sites where native or naturalized host population was widely distributed. There was limited genetic diversity at germplasm evaluation sites, with a large proportion of isolates having identical haplotypes. This contrasts recent pathogenicity results for 78 of the Brazilian isolates that show hot spots of complex races are more common around research stations where host germplasm are tested, but few are found at sites containing wild host populations. For a pathogen in which the same races arise convergently from different genetic backgrounds, this study highlights the importance of using both virulence and selectively neutral markers to understand pathogen population structure.     

Host switching between native and non‐native trees in a population of the canker pathogen Chrysoporthe cubensis from Colombia

The purpose of this study was to test the hypothesis that Chrysoporthe cubensis on native trees in South America could be the source of the pathogen that causes severe stem cankers and often mortality in commercially propagated Eucalyptus trees. This was done by investigating populations originating from two adjacent Eucalyptus (Myrtaceae) plantations in Colombia, and wild Miconia rubiginosa trees (Melastomataceae) growing alongside these stands. Polymorphic microsatellite markers were used to quantify allele sizes in 20 and 39 isolates from the two Eucalyptus stands and 32 isolates from adjacent M. rubiginosa trees. Gene and genotypic diversities were calculated from these data, and population differentiation and assignment tests were performed to ascertain whether the populations were genetically different. Results showed that there were no differences between any of the populations using these techniques, and that they can be treated as a single population. Therefore, the results support the hypothesis that host switching has occurred in C. cubensis in Colombia.     

Diversity and Complexity of Erysiphe graminis f.sp. hordei Collected from Barley Cultivar Mixtures or Barley Plots Treated with a Resistance Elicitor

Powdery mildew populations were analysed to determine the effects of a resistance elicitor and cultivar mixtures on genetic complexity and diversity. Isolations were made from a range of spring barley monocultures and mixtures in a field trial, and characterised for virulence and RAPD profile. In a second trial, isolates were taken from a single mixture from untreated and resistance elicitor-treated areas and from the components of the mixture in monoculture. The mildew population was not only highly heterogeneous for virulence characteristics, but also proved heterogeneous within pathotypes for molecular markers, indicating the major impact of sexual recombination on population structure and the lack of clonal dominance. Various diversity measurements were compared and the value of dissimilarity measurement for revealing genetic distance within a population was highlighted. There was a trend towards increasing complexity as the season progressed, but there was no consistent relationship between cultivar or mixture, disease control treatment, fertiliser treatment, replicate or position in trial, and pathogen genotype. Whilst the resistance elicitor did reduce mildew by 78% in the first trial, and there was no interaction with fertiliser level in its expression, control was substantially less in the second trial. There were no differences between mildew isolates from elicitor and control treatments. It was felt that more effective and consistent resistance elicitors need to be developed before it can be stated that they are unlikely to be eroded by selecting resistant or adapted mildew genotypes.     

Persistent virulence associations in sexual populations of Puccinia coronata

From 1992 to 1999, 18 sets of aecial or uredinial isolates of Puccinia coronata were collected from two sites in Minnesota for analysis of virulence associations. In addition, one set of aecial isolates was collected in Ontario and one in New York. Also, aecial isolates and uredinial isolates collected from scattered locations in Minnesota in 1994 and again in 1995 were bulked for comparison with populations from discrete sites. Isolates were tested for virulence on 26 single- Pc gene oat lines. Virulences to 14 pairs of differential lines were found to be significantly ( P  < 0·05) associated in linkage disequilibrium in at least six of the 24 site × year populations. The significant virulence associations were found among both uredinial and aecial isolates. Linkage disequilibria normally dissipate with repeated generations of sexual reproduction. Finding the same virulence associations repeatedly over years and locations for sexual populations of P. coronata indicates that certain pairs of virulence genes (or avirulence genes) contribute to increased fitness when they occur together, even in the absence of the corresponding resistance genes in the host. Mean virulence complexity did not differ significantly between a site with no known Pc genes in the host population and a site with low frequencies of Pc genes, suggesting little or no selection pressure against unnecessary virulence per se . Means and standard deviations of virulence complexity were similar for aecial and uredinial isolates within sites, which suggests that selection did not strongly favour either heterozygotes or intermediate virulence complexity during uredinial generations.     

Genetic diversity of the annual weed Monochoria vaginalis in southern China detected by random amplified polymorphic DNA and inter-simple sequence repeat analyses

Two molecular genetic screening techniques, RAPD (random amplified polymorphic DNAs) and ISSR (inter-simple sequence repeats), were applied to detect the level and pattern of genetic diversity of Monochoria vaginalis, a common weed of rice fields, in seven populations from southern China. Among these populations, 116 bands were amplified by 18 RAPD primers, of which 34 bands (29.31%) were polymorphic, and 14 ISSR primers produced 111 bands with 87 polymorphic bands (78.38%). Within each population, a relatively low level of genetic diversity was detected by both RAPD and ISSR analyses, with a mean genetic diversity (H) of 0.0348 and 0.0551 respectively. Analysis of molecular variance of the data from the RAPD and ISSR markers detected that the majority of total genetic variation existed among populations (73.50% and 76.70% respectively) and only minor genetic variation within populations (26.50% and 23.30% respectively). Cluster analysis divided the seven populations into two groups, indicating that the genetic relationships among populations have relatively low correlation with their geographical distribution (Mantel test; r = 0.45 and 0.48 respectively). Our results indicated that both RAPD and ISSR markers were effective and reliable for accurately assessing the degree of genetic variation of M. vaginalis. Comparing the two techniques, ISSR markers were more efficient than the RAPD assay. The Mantel test gave r = 0.16, suggesting no correlation between these two molecular markers.     

Comparison of populations of the wilt pathogen Ceratocystis albifundus in South Africa and Uganda

Ceratocystis albifundus is an important fungal pathogen of Acacia mearnsii trees in South Africa. In a previous study, a high level of gene diversity was demonstrated in a South African population of C. albifundus . This, together with the occurrence of the pathogen on native Protea species and its exclusive occurrence in South Africa, led to the hypothesis that C. albifundus is probably native to that country. More recently, C. albifundus has been reported from A. mearnsii in south-western Uganda. The aim of this study was to compare the populations of C. albifundus from Uganda and South Africa based on genetic diversity, population structure and possible gene flow. This was achieved using codominant microsatellite markers developed for the closely related species Ceratocystis fimbriata . Available isolates for comparison were from six different areas of South Africa and six jungle stands in Uganda. Eight of the 11 available markers amplified loci in C. albifundus . Gene diversity was higher in the Ugandan population, but genotypic diversity was greater for the South African isolates. There were no common genotypes between the two populations and they shared only 22% of the total alleles. The populations were genetically isolated from each other and highly substructured within. There was no association between isolates collected from the same geographic locations, and gene flow between the two populations was low. Results suggest that C. albifundus was probably not introduced into Uganda from South Africa but rather that an ancestral population, yet to be discovered, is the source of both populations.     

Genetic Diversity and Structure of the Apiosporina morbosa Populations on Prunus spp

ABSTRACT Populations of Apiosporina morbosa collected from 15 geographic locations in Canada and the United States and three host species, Prunus virginiana, P. pensylvanica, and P. padus, were evaluated using the sequence-related amplified polymorphism (SRAP) technique to determine their genetic diversity and population differentiation. Extensive diversity was detected in the A. morbosa populations, including 134 isolates from Canada and the United States, regardless of the origin of the population. The number of polymorphic loci varied from 6.9 to 82.8% in the geographic populations, and from 41.4 to 79.3% in the populations from four host genotypes based on 58 polymorphic fragments. In all, 44 to 100% of isolates in the geographic populations and 43.6 to 76.2% in populations from four host genotypes represented unique genotypes. Values of heterozygosity (H) varied from 2.8 to 28.3% in the geographic populations and 10.2 to 26.1% in the populations from four host genotypes. In general, the A. morbosa populations sampled from wild chokecherry showed a higher genetic diversity than those populations collected from other host species, whereas the populations isolated from cultivated chokecherry, P. virginiana 'Shubert Select', showed a reduction of genetic diversity compared with populations from wild P. virginiana. Significant population differentiation was found among both the geographic populations (P < 0.05) and populations from different host genotypes (P < 0.02). In the geographic populations, most of populations from cultivated and wild P. virginiana were closely clustered, and no population differentiation was detected except for the populations from Morris, Morden, and Winnipeg, Manitoba, Canada. Furthermore, the populations from P. virginiana in the same geographic locations had higher genetic identity and closer genetic distance to each other compared with those from different locations. Four populations from P. virginiana, P. pensylvanica, and P. padus, were significantly differentiated from each other (P < 0.02), except there was no differentiation between the Shubert Select and wild chokecherry populations (>P> = 0.334). Indirect estimation of gene flow showed that significant restricted gene flow existed between populations from different regions and host species. Gene flow rates (Nm) varied from <1 to 12.5, with higher gene flow rates among population pairs from the same host species (P = 1.000). The analysis of molecular variance revealed that a major genetic variance source came from the genetic variation among isolates within populations regardless of the origin and host genotype of the population. Although some locations had a limited number of isolates, the results of this study clearly showed that the genetic diversity and population differentiation of A. morbosa were closely associated with host genotypes and geographic locations, but mostly with the former.     

Temporal Variation in Setosphaeria turcica Between 1974 and 1994 and Origin of Races 1, 23, and 23N in the United States

ABSTRACT Setosphaeria turcica causes northern leaf blight, an economically important disease of maize throughout the world. Survey collections of S. turcica isolates from 1974 to 1994 provided a unique opportunity to examine temporal diversity in the eastern United States. Two hundred forty-two isolates of S. turcica from maize were studied with random amplified polymorphic DNA (RAPD) markers, mating type, and virulence on maize differential inbred lines with known Ht resistance genes to examine changes over time. One hundred forty-nine RAPD haplotypes were identified. Nearly 20% of haplotypes recurred in more than one year. Race 0 isolates declined in frequency from 83% in 1974 to near 50% in the 1990s, most likely in response to the widespread deployment of Ht1 in commercial maize hybrids. Races 23 and 23N were present in the collection at low levels throughout the study period and were also found among isolates from Virginia in 1957. The frequency of MAT1-2 isolates increased sharply after 1979 and was associated with the emergence of race 1 during the same period. RAPD markers were used to investigate the genetic diversity among a subset of isolates collected in the United States from 1976 to 1982, the period in which this dramatic shift in race frequency occurred. Multilocus haplotypes were not exclusively associated with known races of S. turcica. Based on shared haplotypes and cluster analysis, race 1 isolates share greater similarity with race 0 than with 23 or 23N isolates, indicating race 1 probably evolved from multiple lineages of race 0. Sorghum spp.-infecting isolates share greater similarity with one another than with maize-infecting isolates and represent a distinct subgroup.     

Comparison of Puccinia graminis f.sp. tritici from South America and Europe

Twenty isolates of Puccinia graminis f.sp. tritici from South America were compared with 19 from Europe using virulence, isozymes and random amplified polymorphic DNA (RAPD) markers. The isozyme and virulence patterns for these isolates were also compared with those of 11 isolates representative of the common race clusters in North America. All three types of marker showed a level of similarity between the South American and European isolates comparable with that between isolates from the same continent. The average similarity coefficients between the South American and European isolates were 0.65 for virulence, 0.67 for isozymes, and 0.70 for RAPD markers. Among South American isolates the values were 0.63 for virulence, 0.64 for isozymes and 0.72 for RAPDs. For the South American and European isolates, correlation between the similarity matrices based on RAPDs and on isozyme markers, respectively ( r  = 0.52), was higher than that between the RAPD and virulence matrices ( r  = 0.32) or between isozyme and virulence matrices ( r  = 0.16). The North American isolates had a comparable level of similarity for virulence and isozymes to both the South American and European populations. There was no clear distinction between the South American, North American and European isolates, which is consistent with the hypothesis that these populations may have had a common origin.     

Fine Mapping and Identification of Nucleotide Binding Site/Leucine-Rich Repeat Sequences at the MER Locus in Populus deltoides 'S9-2'

ABSTRACT Melampsora larici-populina is the most damaging leaf pathogen for poplar in Europe. Previous genetic analyses have revealed both qualitative and quantitative resistance to this fungus. As a starting point for positional cloning of the gene or genes conferring qualitative resistance to M. larici-populina races E1, E2, and E3, a local genetic map of the Melampsora resistance (MER) locus was constructed based on amplified fragment length polymorphism (AFLP) markers. Eleven AFLP markers were identified by bulked segregant analysis. These markers were used to identify 17 recombinants at the MER locus, from a total of 512 progenies derived from three interspecific crosses involving the same resistant female parent, Populus deltoides 'S9-2'. The local genetic map covered a 3.4-centimorgan interval encompassing the target locus. Sequence analysis of these AFLP markers revealed similarities to the nucleotide binding site/leucine-rich repeat class of disease resistance genes.     

The threat of wild habitat to scab resistant apple cultivars

Evaluations of plant resistance to pathogens are rarely made using isolates from wild habitats, although the heterogeneity of such habitats may generate pathogen diversity which could be a source of new virulence in cultivated habitats. The aim of this study was to investigate whether scab resistance factors, identified and characterized in apples using isolates of Venturia inaequalis from a cultivated habitat, remained effective against isolates from a wild habitat. Three V. inaequalis core collections originating from the cultivated apple Malus × domestica and from two wild species, M. sieversii and M. sylvestris, were established to maximize pathogen diversity. For each core collection, 10 isolates were inoculated in mixtures onto 51 genotypes from an apple progeny segregating for two qualitative resistance genes and six quantitative resistance loci (QRL). On each apple genotype, isolates that contributed to the scab symptoms were identified within the mixture using microsatellite markers. The most frequently detected isolates were inoculated singly to compare their aggressiveness according to their host origin. The results showed that isolates from a wild habitat were able to infect the susceptible apple genotypes. However, these isolates were never more aggressive than isolates from the cultivated habitat on the resistance factors tested. It can therefore be concluded that the resistance factors used in this study, identified with V. inaequalis isolates from a cultivated habitat, remained effective against isolates from M. sylvestris and M. sieversii.     

Genetic diversity and aggressiveness of Erwinia psidii on Eucalyptus spp. in Brazil

The genetic variability and aggressiveness of Brazilian Erwinia psidii isolates from Eucalyptus spp. was studied and compared with reference isolates from guava (Psidium guajava). Repetitive element sequence (rep)-based PCR markers of 101 isolates from Eucalyptus spp. and five from guava showed that the populations of E. psidii displayed a relatively low genetic variability. No correlation of genetic clustering based on rep-PCR analysis with geographic origin or host of origin was observed, indicating that genome rearrangements associated with adaptation to a particular host were not detected by these molecular markers. A higher genotypic richness was detected in the Mato Grosso do Sul population, probably reflecting a pathogen dissemination associated with the recent expansion in eucalypt plantations. Wilcoxon and ANOVA tests of disease severity data indicated differences in aggressiveness among isolates and an isolate × clone interaction. The area under the disease progress curve (AUDPC) and disease severity for some isolates were significantly different between two susceptible clones tested. Notably, isolate LPF681 from guava was not able to cause disease on a susceptible Eucalyptus urophylla clone, suggesting that some co-evolution between pathogen and host has taken place. The variability in aggressiveness and virulence among isolates of E. psidii observed in this study will be important for the establishment of appropriate screening approaches to select for disease resistance.     

Patterns of Virulence in Natural Populations of Puccinia coronata on Wild Oat in Israel and in Agricultural Populations on Cultivated Oat in the United States

ABSTRACT Crown rust (Puccinia coronata) in indigenous populations of Avena sterilis has been cited as an example of stability of wild pathosystems that consist of natural mixtures of resistance and virulence. This study confirmed that virulence/avirulence polymorphisms in P. coronata on A. sterilis in Israel are highly diverse and that super races do not dominate. Isolates of P. coronata from Israel in 1991 to 1996 were polymorphic for virulence to 35 of 36 differential oat lines with resistance genes from A. sterilis. On average, isolates of P. coronata were more highly virulent to differentials with Pc genes from A. sterilis accessions from Israel than to differentials with Pc genes from other countries. Isolates from Israel also were more virulent on average to 10 additional differentials with Pc genes derived from A. sativa than to differentials with Pc genes from A. sterilis. Frequencies of virulence were usually higher in collections of P. coronata from Israel than in collections from cultivated oat in the United States, even though several of the Pc genes in the differentials have been used extensively in American oat cultivars. Mean virulence complexity of P. coronata from eight regions of Israel was not correlated with the distribution of resistance among collections of A. sterilis from previous surveys in the same areas, probably because pathogen migration between regions within Israel is sufficient to obscure effects of selection locally.     

Genetic Structure and Variation in Aggressiveness in European and Australian Populations of the Grapevine Dieback Fungus, Eutypa lata

Eutypa lata is an ascomycete fungus causing a severe dieback in grapevine. The genetic structure of populations of E. lata from seven regions in Australia, France, Italy and Spain was examined using 20 random amplified polymorphic DNA (RAPD) markers. In some regions, populations were subdivided and a total of 14 samples were analysed. A total of 231 RAPD haplotypes were found among the 240 isolates. Vegetative compatibility testing further demonstrated that isolates of the same haplotype were genetically distinct. Gene diversity was the highest in the population from northern Italy and lowest in the Alsace region in France. Linkage disequilibrium between pairs of putative loci was very low and most of the multilocus analyses were consistent with the hypothesis of random association of the loci. This suggests that random mating occurred in every population and that the sexual stage shapes the genetic structure of E. lata populations in the regions sampled. Only 6% of the total variability was attributable to differences between populations. Nevertheless, significant differences in allele frequency appeared with respect to six RAPD markers indicating some genetic differentiation between populations. This differentiation appeared attributable to differences between the Italian and Spanish populations and the other populations. We thus hypothesize that a restriction of gene flow exists within Europe. The population from Australia was genetically closer to the French and Spanish populations than to that from Italy. Genetic diversity is associated with considerable variation in aggressiveness, which was assessed on cuttings in the greenhouse in six populations. All populations included a range of isolates differing in aggressiveness, but the Italian population seemed to have more isolates with low aggressiveness.     

Analysis of Spanish populations of Ophiostoma ulmi and O. novo-ulmi using phenotypic characteristics and RAPD markers

One hundred and six isolates of the Dutch elm disease (DED) fungi Ophiostoma ulmi and Ophiostoma novo-ulmi were collected from elm trees with symptoms in 15 regions of Spain. Isolates were compared with eight reference strains belonging to O. ulmi and the two subspecies of O. novo-ulmi. The purpose of this study was to assign Spanish isolates to species and subspecies of the DED fungi and to analyse the genetic variability within the Spanish populations of these pathogens. Isolates were examined for their growth rates, colony morphologies and fertility responses and by using random amplified polymorphic DNA (RAPD) markers. Six isolates were identified as O. ulmi , 16 as O. novo-ulmi ssp. novo-ulmi and 78 as O. novo-ulmi ssp. americana . Nei's and Shannon's diversity indices and the upgma dendrogram from RAPD profiles indicated a high level of variation among isolates, probably reflecting post-epidemic status of DED in Spain. Although most isolates were separated into three major clusters representing the three taxa of DED fungi in the RAPD analysis, two isolates from central Spain clustered between O. novo-ulmi ssp. americana and O. novo-ulmi ssp. novo-ulmi , and four isolates from Mallorca clustered between O. ulmi and the group representing O. novo-ulmi ssp. novo-ulmi . Mating tests conducted with these isolates revealed a variety of fertility responses. The novel combinations between molecular profile and fertility reaction suggest that three isolates from Mallorca may be interspecific hybrids of the DED fungi.