AFLP analysis of genetic diversity in populations of <Emphasis Type="Italic">Botrytis elliptica</Emphasis> and <Emphasis Type="Italic">Botrytis tulipae</Emphasis> from the Netherlands

The objective of this study was to assess the genetic diversity and to infer the mode of reproduction of Botrytis elliptica and B. tulipae in the Netherlands. First, three molecular typing methods were compared for their ability to differentiate isolates of B. tulipae, B. elliptica, and B. cinerea. The methods compared were multilocus sequencing, restriction analysis of the ribosomal intergenic spacer (IGS) region, and amplified fragment length polymorphism (AFLP) analysis. AFLP fingerprinting provided the most efficient method to differentiate isolates within each Botrytis species and therefore this method was used for population analyses of B. elliptica and B. tulipae. Isolates of both species were sampled during successive growing seasons in experimental field plots in Lisse and other locations in the Netherlands. Among 174 B. elliptica isolates, 105 genotypes could be discriminated and 87 genotypes were found only once, reflecting high genotypic variation. Clonal genotypes were found only within growing seasons and in one location. Linkage disequilibrium analyses indicated that between 9.4% and 19.3% of the loci in clone-corrected samples were linked. The multilocus association index provided no evidence for random mating. We conclude that sexual recombination occurs in the B. elliptica population. Among the 170 B. tulipae isolates, 25 genotypes could be discriminated and four genotypes were found only once, reflecting a low genotypic variation. Clonal genotypes were frequently found in different growing seasons and different locations. Linkage disequilibrium analyses indicated that between 25.2% and 48.6% of the loci in clone-corrected samples were linked. We conclude that the B. tulipae population is mainly clonal with some recombination.     

Genetic and virulence diversity, and mating type distribution of Togninia minima causing grapevine trunk diseases in Spain

Fifty eight single-spore Togninia minima (anamorph Phaeoacremonium aleophilum) isolates were recovered from grape rootstock wood of plants that showed symptoms of Petri disease and esca from 2001 to 2008 in Spain. These isolates were studied by means of mating type distribution, UP-PCR analysis, and virulence assays. Analysis of clone-corrected data sets showed equal frequencies of both mating types in the entire Spanish population, in the Ciudad Real region, at inter-vineyard and intra-vine spatial scales; while unequal mating type distribution was detected in Valencia and Zaragoza regions, at intra-vineyard and intra-vine spatial scales. This is the first study on distribution of T. minima mating types on spatial scales varying from vineyards to regions. A total of 49 polymorphic UP-PCR markers were obtained using seven UP-PCR primers. Four optimal clusters were inferred with Bayesian structure and multivariate analyses from the UP-PCR data. The high number of unique genotypes observed within the Spanish population, combined with a near-equal distribution of mating types, suggested that sexual reproduction probably does occur. However, based on allele distribution and frequency, each of the three subpopulations appeared to be evolving independently. Gene and genotype diversities across the subpopulations were similar and ranged from 0.24 to 0.27 and from 0.27 to 0.37, respectively. The detection of genetically identical isolates within and among subpopulations indicates that an asexual reproductive component should not be excluded. Contrast analysis among groups defined by UP-PCR analyses showed no significant differences in the virulence of T. minima isolates.     

Population Genetic Structure of Tapesia acuformis in Washington State

ABSTRACT Eyespot of wheat is caused by Tapesia yallundae and T. acuformis. Historically, T. yallundae has been considered the more important causal agent of the disease in Washington state and consists of a large homogeneous population with a genetic structure consistent with both sexual and asexual reproduction. T. acuformis has increased significantly in Washington in the past 10 years and apothecia were found recently under natural field conditions, indicating that T. acuformis may have a more important role in eyespot of wheat than previously was thought. To determine the genetic structure of T. acuformis in Washington, 141 single conidial isolates were sampled from four subpopulations in the eastern wheat-growing region of the state. Isolates were scored for mating type and six amplified fragment length polymorphism markers. All markers segregated in a 1:1 ratio and were determined to be unlinked based on genetic analysis of 24 progeny from an in vitro cross. No significant differences in allele frequencies (0.127 < P < 0.809) were found among individual loci across the four subpopulations and over all loci based on contingency table analysis of the log-likelihood ratio statistic G(2). Likewise, no overall differences between subpopulations were detected using the population differentiation statistic theta (theta = -0.004, P = 0.537). Random mating could not be rejected within each subpopulation or for the combined data using clone-corrected data sets based on (i) 1:1 ratio of mating-type, (ii) multilocus gametic disequilibrium analyses (index of association), (iii) phylogenetic analyses (parsimony tree length permutation test), and (iv) genotypic diversity analyses. T. acuformis has a genetic structure similar to that of sympatric populations of T. yallundae in Washington, with both sexual and asexual reproduction contributing to the structuring of this species.     

Contrasting genetic structure between Magnaporthe grisea populations associated with the golf course turfgrasses Lolium perenne (perennial ryegrass) and Pennisetum clandestinum (kikuyugrass)

Gray leaf spot (GLS) disease of perennial ryegrass (Lolium perenne) and kikuyugrass (Pennisetum clandestinum) in golf courses in California was first noted in 2001 and 2003, respectively, and within 5 years had become well established. The causal agent of the disease is the fungus Magnaporthe grisea, which is known to consist primarily of clonal lineages that are highly host specific. Therefore, our objective was to investigate host specificity and population dynamics among isolates associated primarily from perennial ryegrass and kikuyugrass since the disease emerged at similar times in California. We also obtained isolates from additional hosts (tall fescue, St. Augustinegrass, weeping lovegrass, and rice) and from the eastern United States for comparative purposes. A total of 38 polymorphic amplified fragment length polymorphism makers were scored from 450 isolates which clustered by host with high bootstrap support (71 to 100%). Genetic structure between kikuyugrass and perennial ryegrass isolates differed significantly. Isolates from kikuyugrass were genotypically diverse (n = 34), possessed both mating types, and some tests for random mating could not be rejected, whereas isolates from perennial ryegrass were less genotypically diverse (n = 10) and only consisted of a single mating type. Low genotypic diversity was also found among the other host specific isolates which also only consisted of a single mating type. This is the first study to document evidence for the potential of sexual reproduction to occur in M. grisea isolates not associated with rice (Oryza sativa). Moreover, given the significant host specificity and contrasting genetic structures between turfgrass-associated isolates, the recent emergence of GLS on various grass hosts in California suggests that potential cultural practices or environmental changes have become conducive for the disease and that the primary inoculum may have already been present in the state, despite the fact that two genotypes associated with perennial ryegrass and St. Augustinegrass in California were the same as isolates collected from the eastern United States.     

Virulence profile and genetic structure of a North Dakota population of Pyrenophora teres f. teres, the causal agent of net form net blotch of barley

A Pyrenophora teres f. teres population in North Dakota was analyzed for virulence variation and genetic diversity using 75 monospore isolates that were collected across a 4-year period (2004 to 2007) from two North Dakota State University agricultural experiment stations at Fargo and Langdon. Pathogenicity tests by inoculation onto 22 barley differential lines at seedling stage revealed 49 pathotypes, indicating a wide range of pathogenic diversity. Two-way analysis of variance of disease ratings revealed a significant difference in the virulence among isolates and in the resistance among barley lines, as well as in the interactions between the two. 'CI5791', 'Algerian', and 'Heartland' were three barley lines showing a high level of seedling resistance to all North Dakota isolates tested; however, many previously reported resistance genes have been overcome. Forty multilocus genotypes were identified from this set of isolates by genotyping at 13 simple-sequence repeat loci. High percentages of clonal cultures were detected in the samplings from 2005 and 2007 in Fargo and 2005 in Langdon. Using a clone-corrected sample set, the mean gene diversity (h) was estimated to be 0.58, approximately the same for both locations. The calculated Wright's F(ST) value is small (0.11) but was significantly >0, indicating a significant differentiation between the Fargo and Langdon populations. In the gametic disequilibrium test, only 3 of 78 possible pairwise comparisons over all isolates showed significant (P < 0.05) nonrandom association, suggesting a random mating mode. Our results suggest that the populations from the two locations are derived from a common source and undergo frequent recombination. This research provides important information for barley breeders regarding development and deployment of cultivars with resistance to net form net blotch in this region.     

Phenotypic and genotypic diversity of Phytophthora infestans populations in Scotland (1995–97)

In a survey of Scottish potato late blight ( Phytophthora infestans ) populations from 1995 to 1997, nearly 500 isolates were collected from over 80 disease outbreaks in commercial potato crops and gardens/allotments. The isolates were characterized by mating type, resistance to the fungicide metalaxyl and almost 300 were examined by DNA-based AFLP fingerprinting. These data were examined alongside cropping details to determine the population structure in the context of existing disease management strategies. A1 and A2 mating type isolates were present in both commercial potato crops and gardens or allotments although they coexisted more frequently in the latter sites. One-fifth of the isolates collected were of the A2 mating type and the frequency was similar over the 3 years and amongst sites. In 1995 the proportions of isolates that were sensitive and resistant to metalaxyl were equal (∼40%) but, over the following 2 years, the frequency of resistant isolates decreased and that of intermediate isolates increased. The mating type response to metalaxyl differed markedly, with 52% of A1 and only 5% of A2 isolates being resistant. Considerable molecular diversity was observed, with over half of the isolates having unique AFLP patterns. Analysis of the molecular and phenotypic data revealed a broad clustering of the population into three groups. Many factors point to an A2 population restricted by its sensitivity to phenylamides. The majority of the A2 isolates were found in a single AFLP group, but the presence of mixed mating type samples, an increasing frequency of isolates of intermediate metalaxyl resistance and the extent of the AFLP diversity suggest occasional sexual recombination, and thus gene flow, between groups.     

Genetic diversity and structure of Hemileia vastatrix populations on Coffea spp.

Coffee leaf rust is the most limiting disease for coffee cultivation in Brazil. Despite its importance, relatively little is known about the genetic diversity of Hemileia vastatrix, the rust causal agent. In this work, the DNA from 112 monopustule isolates from different geographic locations and coffee genotypes were analysed by amplified fragment length polymorphisms (AFLP). The objectives were to assess the influence of the host and geographic origin on the diversity and population differentiation in H. vastatrix. The fungal population showed a low level of genotypic diversity. Gene diversity (h) was 0·027 and the hypothesis of random mating in the total population was rejected, but evidence for recombination was found for two subpopulations (São Paulo and Paraná). The analysis of molecular variance revealed that 90% of the genetic distribution of the pathogen occurs among isolates within the subpopulation (states or host of origin). There was no correlation between geographic and genetic distance (r = ?0·024, P = 0·74), which together with the high number of migrants and the low degree of differentiation in populations of H. vastatrix, is consistent with the fact that the inoculum is probably easily dispersed by wind over long distances, allowing dispersal of the pathogen among coffee growing areas in Brazil. Therefore, it is difficult to predict the durability of resistant sources to coffee rust. The recommendation for the breeding programmes is thus to incorporate multigenic resistance as a control strategy.     

Investigating the Spatiotemporal Genetic Structure of Phytophthora capsici in Michigan

ABSTRACT Phytophthora capsici isolates were recovered from pepper and cucurbit hosts at seven locations in Michigan from 1998 to 2000. Isolates were characterized for compatibility type (CT), mefenoxam sensitivity (MS), and amplified fragment length polymorphism (AFLP) marker profiles. In total, 94 AFLP bands were resolved. Individual populations were highly variable. Within populations, 39 to 49% of the AFLP bands were polymorphic and estimated heterozygosities ranged from 0.16 to 0.19. Of the 646 isolates fingerprinted, 70% (454) had unique AFLP profiles. No clones were recovered between years or locations. Pairwise F statistics (Phi(ST)) between populations from different locations ranged from 0.18 to 0.40. A tree based on unweighted pair-group method with arithmetic average cluster analysis indicates discrete clusters based on location. Isolates from the same location showed no clustering based on the year of sampling. Analysis of molecular variance partitioned variability among (40%) and within populations (60%). The overall estimated Phi(ST) was 0.34 (SD = 0.03). A1/A2 CT ratios were approximately 1:1, and MS frequencies were similar between years for the two locations sampled over time. These data suggest that P. capsici persists in discrete outcrossing populations and that gene flow among locations in Michigan is infrequent.     

Genetic structure of <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> populations isolated from maize in Argentina

Fusarium verticillioides (sexual stage Gibberella moniliformis) is a common fungal pathogen of maize worldwide that also produces fumonisin mycotoxins. Populations of this fungus can be diverse with respect to neutral and selectable genetic markers. We used vegetative compatibility groups (VCGs) and amplified fragment length polymorphisms (AFLPs) to evaluate the genetic structure of three F. verticillioides populations from commercial maize fields in Argentina. Based on work with similar populations from outside South America, we expected individuals within the populations to be genetically diverse, that genotypic variation would be distributed in a manner consistent with random mating, and that populations from different locations would be genetically indistinguishable from one another. We analysed 62 AFLP loci for 133 fungal isolates. All three populations were genotypically diverse but genetically similar and potentially part of a larger, randomly mating population, with significant genetic exchange occurring between the three subpopulations. There was no evidence for linkage disequilibrium at P = 0.05. The low values of G _ST , the lack of frequent private alleles, and the lack of a systemic pattern of linkage disequilibrium all suggest that sexual reproduction is sufficiently common in F. verticillioides and that the dispersal of strains is sufficiently efficient for the population of F. verticillioides in the main maize growing region to be a single randomly mating population with no detectable genetic subdivision. Thus differences in disease and/or toxin production observed in this region are best attributed to differences other than the genetic composition of the population.     

Genetic diversity of Australian Fusarium graminearum and F. pseudograminearum

Genotypic diversity in Fusarium pseudograminearum and F. graminearum from Australia and the relationship between diversity and pathogen aggressiveness for head blight and/or crown rot of wheat were examined. Amplified fragment length polymorphism (AFLP) analysis revealed a high level of genotypic diversity within each species. Sixty-three of the 149 AFLP loci were significantly different between the two species and 70 of 72 F. pseudograminearum and 56 of 59 F. graminearum isolates had distinct haplotypes. When head blight and crown rot severity data from a recently published work on isolates representing the entire range of aggressiveness were used, only the genotypic diversity of F. pseudograminearum was significantly associated with its aggressiveness for the two diseases. Cluster analyses clearly demonstrated the polyphyletic structures that exist in both pathogen populations. The spatial diversity within F. graminearum was high within a single field, while frequent gene flow ( N _m ∼ 14) and a low fixation index ( G _st = 0·03) were recorded among F. pseudograminearum isolates from the adjacent states of New South Wales and Queensland. The differences in population structure between the heterothallic F. pseudograminearum (teleomorph G. coronicola ) and the homothallic F. graminearum (teleomorph G. zeae ) were not as pronounced as expected given their contrasting mating systems. Neither species was panmictic or strictly clonal. This points to sexual recombination in F. pseudograminearum , suggesting that ascospores of G. coronicola may also play a role in its biology and epidemiology.     

Genetic Variability of Canadian Populations of the Sapstain Fungus Ophiostoma piceae

ABSTRACT Genetic diversity was studied in seven Canadian populations of Ophiostoma piceae, the most prevalent sapstain fungus in Canadian softwoods. A total of 239 single-spore isolates were recovered following a systematic survey of sapstain fungi in logs and lumber at seven selected sawmills in six Canadian provinces (British Columbia, Alberta, Saskatchewan, Ontario, Québec, and New Brunswick). Sampling was carried out on five commercially important softwood species: balsam fir (Abies balsamea), white spruce (Picea glauca), black spruce (Picea mariana), jack pine (Pinus banksiana), and lodgepole pine (Pinus con-torta var. latifolia). The A and B mating types occurred at equal frequency (MAT A/ MAT B = 1.00:1.13) over all populations. Pseudo-allelic frequencies were estimated at each of 24 putative genetic loci by scoring for presence or absence of random amplified polymorphic DNA fragments generated by five primers. A total of 237 haplotypes were found among the 239 isolates, revealing a high level of genotypic diversity among isolates. Total gene diversity (H(T) = 0.414) was mostly attributable to diversity within populations (H(S) = 0.369). Thus, only 11.2% of the total variability was attributable to frequency differences among populations. An analysis of molecular variance revealed that most genetic variability occurred within subpopulations within mills (84.3%; P < 0.001), whereas low but statistically significant levels of genetic differentiation were also observed among subpopulations within populations (5.4%; P < 0.001) and among populations (10.3%; P < 0.001). Estimates of Nei' genetic distances were not correlated with geographic distances among sampling locations (r = -0.092; P = 0.310), although principal component analysis indicated that subpopulations located east of Saskatchewan were grouped on the same side of the second principal component axis. Overall, results suggest moderate genetic differentiation of O. piceae in Canada, which is consistent with the observation that sexual reproduction is frequently observed in this fungus.     

Population Analysis of Fusarium graminearum from Wheat Fields in Eastern China

ABSTRACT Wheat heads showing symptoms of Fusarium head blight were collected from four commercial fields in Zhejiang Province, China, an area where epidemics occur regularly. A total of 225 isolates were subjected to population-level analyses using restriction fragment length polymorphism (RFLP) as markers. Diagnostic RFLP markers established that all isolates belonged to Fusarium graminearum lineage 6. Nine polymorphic probes were hybridized to all isolates, resulting in 65 multilocus RFLP haplotypes (MRH). Probing with the telomeric clone pNla17, which reveals differences among isolates in the hypervariable subtelomeric region, differentiated the 65 MRH further into 144 clones. Mean gene diversity for the four field populations was similar, ranging from H = 0.306 - 0.364 over the nine RFLP loci for clone-corrected data. High levels of gene flow were inferred from a low level of population subdivision among all field populations, indicating that they were part of the same population. Pairwise linkage disequilibrium measures did not unequivocally support a random mating population, because one-third of locus pairs were significantly different from the null hypothesis of no-association between alleles. We speculate therefore that sexual recombination may not be frequent and that high levels of genotypic diversity may be maintained by relatively low selection pressure acting on a highly diverse population.     

Population structure and mating system of Ascochyta rabiei in Tunisia: evidence for the recent introduction of mating type 2

The population structure of Ascochyta rabiei (teleomorph: Didymella rabiei ) in Tunisia was estimated among five populations sampled from the main chickpea growing regions using simple sequence repeat markers (SSR) and a mating type ( MAT ) marker. Mating type 2 isolates ( MAT1-2 ) had reduced genetic and genotypic diversity relative to mating type 1 isolates ( MAT1-1 ). This result, coupled with previous observations of lower overall frequency and restricted geographical distribution of MAT1-2 in Tunisia, and recent (2001) observation of the sexual stage, support the hypothesis of a recent introduction of MAT1-2 . Despite the presence of both mating types in Nabeul, Kef and Jendouba, the hypothesis of random mating was rejected in these locations with multilocus gametic disequilibrium tests. Highly significant genetic differentiation ( θ  = 0·32, G _ST = 0·28, P  < 0·001) was detected among populations and genetic distance and cluster analyses based on pooled allele frequencies revealed that populations from Nabeul and Kef were distinct from those in Beja, Bizerte and Jendouba. More than 70% of total gene diversity ( H _T = 0·55) detected was attributable to variation within populations compared to 28% among populations. This result, coupled with the occurrence of private alleles in each population, suggests that gene flow is currently limited among populations, even those separated by short geographic distances. The presence of two main genetic clusters was confirmed using Bayesian model-based population structure analyses of multilocus genotypes (MLGs) without regard to geographic origin of samples. The presence of MAT1-2 isolates in both clusters suggests at least two independent introductions of MAT1-2 into Tunisia that are likely to be the result of importation and planting of infected chickpea seeds.     

Population Structure of Botryosphaeria dothidea from Pistachio and Other Hosts in California

ABSTRACT Genetic diversity was investigated among California populations of Botryosphaeria dothidea, causal agent of Botryosphaeria panicle and shoot blight of pistachio, with random amplified polymorphic DNA (RAPD) and microsatellite-primed polymerase chain reaction (MP-PCR). We surveyed 120 isolates, 112 of which originated from the California Central Valley and included pistachio isolates (n = 52) and isolates from other plant species (n = 60). Out-group isolates (n = 8) were obtained from pistachio in Greece. There was a strong correlation (r = 0.99; P < 0.0001) between the RAPD- and MP-PCR dissimilarity data sets. Little genetic variation (haplotypic diversity [Hs] < 0.002) was detected among B. dothidea isolates collected from central and southern California pistachio plantings. We observed relatively high diversity for isolates from a northern California pistachio orchard (Hs = 0.0146), where the disease was first diagnosed, and from the Chico U.S. Department of Agriculture Germ Plasm Repository (Hs = 0.0726), where the first pistachio trees were planted in California in 1929. Isolates obtained from other hosts, especially those associated with the rare occurrence of the sexual stage of this fungus, showed the highest levels of diversity (Hs = 0.1689). Thirty-eight pistachio isolates (73.1%) had DNA fingerprints identical to 28 pycnidiospore-derived isolates (56.0%) obtained from other host species. Greenhouse inoculations demonstrated that all isolates obtained from other hosts were capable of infecting pistachio and produced characteristic disease symptomology. Thus, California populations of B. dothidea from pistachio are, for the most part, genetically uniform, with the sexual stage rare to absent. However, the rare occurrence of the sexual stage of B. dothidea on other hosts, and more importantly, the capacity of these isolates to infect pistachio, indicate that other host species may serve as sources of inoculum and genetic variation.     

Comparison of RAPD and AFLP Marker Analysis as a Means to Study the Genetic Structure of Botrytis cinerea Populations

To assess the genetic relationships of Botrytis cinerea populations in Almería (Spain), 44 isolates of B. cinerea, collected from six commercial greenhouses (subpopulations), were analysed by Random Amplified Polymorphic DNA (RAPD) and amplified-fragment length polymorphism (AFLP). Polymorphisms were more frequently detected per primer with AFLP than with RAPD (16 compared to 4). However, RAPD detected polymorphisms more frequently per loci than AFLP (56% compared to 32%). The analysis of population structure revealed that the genetic diversity within subpopulations (H_S) accounted for 96% of the total genetic diversity (H_T) , while genetic diversity among subpopulations represented only 4% of the total diversity, independently of whether they were analysed with RAPD or AFLP markers. The relative magnitude of gene differentiation between subpopulations (G_ST) and the estimate of the number of migrants per generation (N_m) averaged similar values when estimated with RAPD or AFLP markers (0.039 and 0.036, or 12.32 and 13.39, respectively). The results obtained in dendrograms were in accordance with the gene diversity analysis. However, the diversity of B. cinerea was higher when analysed by RAPD than with AFLP. In these cases, the isolates could not be grouped by greenhouse or fungicide resistance (except those sensitive to carbendazim and resistant to procymidone). Both the RAPD and AFLP technologies are suitable for studies of genetic structure of B. cinerea populations, although RAPD generated more polymorphisms per loci than AFLP, and provided a better explanation of the genetic relationships between isolates.     

Application of amplified restriction fragment length polymorphism for genetic characterization of colletotrichum pathogens of alfalfa

ABSTRACT Amplified restriction fragment length polymorphism (AFLP) was used to assess the levels of genomic variations among species and isolates of the genus Colletotrichum. Our objective was to characterize at the molecular level two alfalfa pathogens, isolates Arl-NW and 57RR, which are unusually aggressive to anthracnose-resistant alfalfa cultivars and whose taxa has been uncertain based on morphological criteria. The fingerprint patterns obtained were complex but did enable us to place these two isolates within the species C. trifolii and C. gloeosporioides, respectively. The diversity detected with AFLP among and within Colletotrichum species from alfalfa and other crops corroborated their published taxonomy based on morphology, ribosomal DNA sequence, and random amplified polymorphic DNA analyses. Similarity matrices generated with three primer pairs were highly correlated and, thus, were combined to determine the similarity among the fungal species and isolates that were analyzed. Analysis of the data generated with each of the primer pairs individually and application of either distance or parsimony methods supported the placement of these two isolates. The parsimony method of data analysis was more confirmatory in the placement of Phoma medicaginis as an out-group than the distance method, using either simple matching or Jaccard's coefficients to generate the similarity matrices. Our conclusion is that the AFLP technique will be useful for identification of individual isolates within complex genera such as Colletotrichum because of its ability to generate large numbers of polymorphisms and the consistency of polymerase chain reaction amplification.     

Virulence and Molecular Diversity in Cochliobolus sativus

ABSTRACT Spot blotch, caused by the fungal pathogen Cochliobolus sativus, is an important disease of barley in many production areas of the world. To assess genetic diversity in this pathogen, a worldwide collection of C. sativus isolates was evaluated for virulence on barley and DNA polymorphism. Three pathotypes (0, 1, and 2) were identified among the 22 isolates tested in this study and the 36 isolates characterized previously on three barley differentials (ND5883, Bowman, and NDB112) that differ in their resistance to C. sativus. Pathotype 2, which exhibits high virulence on cv. Bowman, was only found in North Dakota, whereas the other two pathotypes occurred in many other regions of the world. Genetic diversity of the 58 C. sativus isolates, together with isolates of three related pathogenic Cochliobolus spp. (C. heterostrophus, C. carbonum, and C. victoriae) was analyzed using amplified fragment length polymorphism (AFLP) markers. A total of 577 polymorphic AFLP markers were recorded among the 70 isolates of the four Cochliobolus spp. using eight primer combinations. Cluster analysis revealed distinct groups corresponding to the four different species, except in one case where race 0 of C. carbonum was placed in an outgroup that may belong to a different species. In C. sativus, 95 polymorphic AFLP markers were detected with the eight primer pairs used, and each isolate exhibited a unique AFLP pattern. Allelic diversity in the pathotype 2 group was lower (0.10) than in the pathotype 0 (0.23) and pathotype 1 (0.15) groups, indicating that pathotype 2 may have arisen more recently. Cluster analysis did not reveal a close correlation between pathotypes and AFLP groups, although two AFLP markers unique to pathotype 2 isolates were identified. This low correlation suggests that genetic exchange may have occurred through parasexual recombination in the fungal population. Some isolates collected from different regions of the world were clustered into the same AFLP group, suggesting that migration of the fungal pathogen around these regions has occurred.     

Indirect evidence for sexual reproduction in Cercospora beticola populations from sugar beet

Cercospora beticola is the main causal agent of cercospora leaf spot on sugar beet and has a large negative impact on the yield and quality of sugar beet production worldwide. Previous studies have shown that both mating type idiomorphs of C. beticola are present in natural populations, suggesting that C. beticola is heterothallic and may be reproducing sexually. Cercospora beticola isolates are diverse in the morphology of their conidia, onset of disease symptoms and fungicide resistance. To find the source of this diversity and to determine if sexual reproduction occurs in this fungus, C. beticola populations were collected from Western Europe, Iran and New Zealand. The mating types of these isolates were determined and AFLP analyses were used to study the genetic diversity in these populations. The mating type ratios did not deviate significantly from a 1:1 ratio in most of the populations and AFLP analyses showed high levels of genetic variation within and between the populations, with 86·4% of the isolates having unique genotypes. All populations were in significant linkage disequilibrium but levels of disequilibrium were low, and loci from only one primer pair were in significant gametic equilibrium in populations from the Netherlands and Italy. From these results there is the possibility that C. beticola reproduces sexually. High levels of gene flow among the samples from Europe demonstrated a single panmictic European population. This study confirms C. beticola to be a genetically highly diverse species, supporting the assumption that some populations are reproducing sexually.     

The recent history of Puccinia striiformis f.sp. tritici in Denmark as revealed by disease incidence and AFLP markers

Disease observations and amplified fragment length polymorphism (AFLP) markers were used to study recent developments in the Puccinia striiformis f.sp. tritici population in Denmark. The fungus appeared spontaneously at 10 locations in Denmark in 1997 after it was not observed under natural conditions in 1996. The pattern of disease development and prevailing winds suggested that the fungus reappeared by airborne spores from the south or west. In 1998, disease incidence was more evenly distributed throughout the country. Forty-eight single lesion isolates were collected from most crops where the disease was observed in these years; all except one from 1997 belonged to two pathotypes that were not previously detected in the country, and both possessed the newly discovered Yr17 virulence. The isolates were characterized with AFLP markers together with 28 isolates representing eight of 13 pathotypes observed prior to 1996. Initial screening of 240 Pst I/ Mse I AFLP primer combinations on four isolates showed that a primer combination, on average, revealed 0·4 polymorphisms between any isolate pair. A selection of 21 primer combinations resulted in 28 AFLP markers, which revealed 16 AFLP phenotypes among all 76 isolates. The two Yr17- virulent pathotypes consisted of three AFLP phenotypes, which were observed in both 1997 and 1998; the two most frequent AFLP phenotypes occurred at most sampling locations and often within the same crop. AFLP diversity was larger among samples collected prior to 1996, and also in this period most AFLP phenotypes were observed at different sampling locations. These results are consistent with the features of an entirely asexually reproducing pathogen dispersed by aerial spores across large areas.     

High Genetic Similarity Among Populations of Phaeosphaeria nodorum Across Wheat Cultivars and Regions in Switzerland

ABSTRACT Phaeosphaeria nodorum was sampled from nine wheat fields across a 30-km transect representing three geographical regions in Switzerland to determine the scale of genetic differentiation among subpopulations. Three different wheat cultivars were sampled three times to determine whether differences in host genotype correlated with differences among corresponding pathogen populations. Seven restriction fragment length polymorphism (RFLP) loci and one DNA fingerprint were assayed for each of the 432 isolates in the collection. DNA fingerprints differentiated 426 unique genotypes. Though absolute differences were small, five RFLP loci exhibited significant differences in allele frequencies across the nine sub-populations. Gene diversity within all subpopulations was high (H(T) = 0.51), but only 3% of the total genetic variation was distributed among the nine subpopulations. When subpopulations were grouped according to geographical region or host cultivar, less than 1% of the genetic variation was distributed among groups, suggesting widespread gene flow and the absence of pathogen adaptation to specific wheat cultivars. Tests for gametic equilibrium within subpopulations and across the entire Swiss population supported the hypothesis of random mating.