Comparative assessment of SSR and SNP markers for inferring the population genetic structure of the common fungus Armillaria cepistipes.

During the last years, simple sequence repeats (SSRs, also known as microsatellites) and single-nucleotide polymorphisms (SNPs) have become the most popular molecular markers for describing neutral genetic variation in populations of a wide range of organisms. However, only a limited number of studies has focused on comparing the performance of these two types of markers for describing the underlying genetic structure of wild populations. Moreover, none of these studies targeted fungi, the group of organisms with one of the most complex reproductive strategies. We evaluated the utility of SSRs and SNPs for inferring the neutral genetic structure of Armillaria cepistipes (basidiomycetes) at different spatial scales. For that, 407 samples were collected across a small (150 km2) area in the Ukrainian Carpathians and a large (41 000 km2) area in the Swiss Alps. All isolates were analyzed at 17 SSR loci distributed throughout the whole genome and at 24 SNP loci located in different single-copy conserved genes. The two markers showed different patterns of structure within the two spatial scales studied. The multi-allelic SSR markers seemed to be best suited for detecting genetic structure in indigenous fungal populations at a rather small spatial scale (radius of ~50-100 km). The pattern observed at SNP markers rather reflected ancient divergence of distant (~1000 km) populations that in addition are separated by mountain ranges. Despite these differences, both marker types were suitable for detecting the weak genetic structure of the two A. cepistipes populations investigated.

Invasion genetics of the chestnut blight fungus Cryphonectria parasitica in Switzerland.

Cryphonectria parasitica is the best-known example of an invasive forest pathogen in Europe. In southern Switzerland, chestnut blight was first reported in 1948 whereas, north of the Alps, it did not appear until the 1980s. Between 1995 and 2008, we sampled 640 C. parasitica isolates from nine populations south of the Alps and nine north of the Alps. Twelve historical isolates, collected between 1950 and 1972 in the south, were obtained from our collection. All 652 isolates were screened at 10 microsatellite loci to test for the existence of divergent genetic pools and to infer possible origins of haplotypes. In total, 52 haplotypes were identified. Structure software analysis indicated that 43 haplotypes (including all historical haplotypes) belonged to a main cluster, 6 haplotypes belonged to a different cluster, and 3 haplotypes had an intermediate allele pattern. All newly founded populations in northern Switzerland were initiated by one or just a few haplotypes from the main cluster, which probably came directly from the populations south of the Alps. Subsequently, genetic diversity increased through mutations, sexual reproduction, or new migrations. The highest increase in diversity was observed in populations where haplotypes from different genetic pools were encountered.

Migration patterns of the emerging plant pathogen Phytophthora ramorum on the West Coast of the United States of America.

Phytophthora ramorum (oomycetes) is the causal agent of sudden oak death and ramorum blight on trees, shrubs, and woody ornamentals in the forests of coastal California and southwestern Oregon and in nurseries of California, Oregon, and Washington. In this study, we investigated the genetic structure of P. ramorum on the West Coast of the United States, focusing particularly on population differentiation potentially indicative of gene flow. In total, 576 isolates recovered from 2001 to 2005 were genotyped at 10 microsatellite loci. Our analyses of genetic diversity and inferences of reproductive mode confirm previous results for the Oregon and California populations, with the strong majority of the genotypes belonging to the NA1 clonal lineage and showing no evidence for sexual reproduction. The high incidence of genotypes shared among populations and the lack of genetic structure among populations show that important large-scale, interpopulation genetic exchanges have occurred. This emphasizes the importance of human activity in shaping the current structure of the P. ramorum population on the West Coast of the United States.

Genetic structure of an expanding Armillaria root rot fungus (Armillaria ostoyae) population in a managed pine forest in southwestern France.

The Landes de Gascogne forest (southwestern France) is the largest maritime pine (Pinus pinaster) plantation in Europe. Armillaria root disease (Armillaria ostoyae) has been reported since the early 1920s in the coastal area (western sector), but its incidence over the last 20 years has increased in the eastern sector. We investigated the genetic structure of the A. ostoyae population in this forest, focusing particularly on geographical differentiation potentially indicative of disease expansion in this area. In total, 531 isolates obtained from mycelial fans on symptomatic trees or undecayed stumps in 31 different disease foci were genotyped at five microsatellite loci. In 20 of these disease foci, a single genotype dominated, reflecting a predominantly clonal local spread of A. ostoyae. By contrast, at the regional scale, A. ostoyae probably spreads mostly via basidiospores (sexual spores), as no genotype common to several disease foci was identified. The absence of a clear pattern of isolation by distance may indicate either substantial gene flow or stochastic colonisation independent of spatial distance. The gradient of genetic diversity from the coast inwards and the greater genetic divergence of the eastern disease foci are consistent with the expansion of the A. ostoyae population from the coast eastwards.

Reconstruction of the Sudden Oak Death epidemic in California through microsatellite analysis of the pathogen Phytophthora ramorum.

The genetic structure of the clonally reproducing Sudden Oak Death (SOD) pathogen in California was investigated using seven variable microsatellites. A total of 35 multilocus genotypes were identified among 292 samples representative of populations from 14 forest sites and of the nursery trade. amova indicated significant genetic variability both within (44.34%) and among populations (55.66%). Spatial autocorrelation analyses indicated that Moran's index of similarity reached a minimum of 0.1 at 350 m, increased to 0.4 at 1500 m and then decreased to zero at 10 km. These results suggest a bimodal pattern of spread, with medium range dispersal (1500-10,000 m) putatively attributed to the presence of strong winds. Lack of genetic structure was identified for three groups of populations. One group notably included the nurseries' population and two forest populations, both linked to early reports of the pathogen. A neighbour-joining analysis based on pairwise Phi(ST) values indicated that the clade inclusive of the nurseries' populations is basal to all California populations. A network analysis identified three common genotypes as the likely founders of the California infestation and proposes a stepwise model for local evolution of novel genotypes. This was supported by the identification in the same locations of novel genotypes and of their 1- or 2-step parents. We hypothesize that the few undifferentiated population groups indicate historical human spread of the pathogen, while the general presence of genetically structured populations indicates that new infestations are currently generated by rare medium or long-range natural movement of the pathogen, followed by local generation of new genotypes.

Saprophytic Activity and Sporulation of Cryphonectria parasitica on Dead Chestnut Wood in Forests with Naturally Established Hypovirulence.

ABSTRACT Sustainable biological control of the chestnut blight fungus Crypho-nectria parasitica with hypovirulence depends on the production and dissemination of hypovirus-infected propagules of the pathogen. We investigated the ability of C. parasitica to sporulate and produce hypo-virus-infected spores on recently dead chestnut wood in coppice stands in southern Switzerland where hypovirulence has been naturally established. The number and type (active, inactive, or none) of cankers was assessed on experimentally cut and stacked stems, firewood stacks, and natural dead wood. Hypovirus-free and hypovirus-infected strains readily survived for more than 1 year in the chestnut blight cankers of the stacked stems. Sporulation of C. parasitica was observed on the surface of preexisting inactive and active cankers, as well as on newly colonized bark areas and was significantly more abundant than on comparable cankers on living stems. On all types of dead wood, we observed more stromata with perithecia than with pycnidia; however, a large proportion of the stromata was not differentiated. All perithecia examined yielded only hypovirus-free ascospores. The incidence of pycnidia that produced hypovirus-infected conidia ranged from 5% on natural dead wood to 41% on the experimental stacks. The mean virus transmission rate into conidia was 69%. Our study demonstrates a considerable saprophytic activity of C. parasitica on recently dead chestnut wood and supports the hypothesis of a role of this saprophytic phase in the epidemiology of hypovirulence.

[Headache in childhood: clinico-epidemiologic study. Preliminary communication: The headache risk]. / La cefalea dell'età evolutiva: studio clinico epidemiologico. Comunicazione preliminare: Il rischio cefalalgico.

After a review of the attempt to relate headache risk factors to the Periodic Syndrome, preliminary data from a clinical epidemiological survey of 980 children (449 M and 531 F aged 4-14 with Idiopathic Headache are reported. The importance of HRFs as predictors of headache is emphasised as is the need to start prophylaxis before headache attacks begin.

[Enuresis: clinico-epidemiologic study. Preliminary communication: its relation to essential headache]. / L'enuresi: indagine clinico-epidemiologica. Comunicazione preliminare: i suoi rapporti con la cefalea essenziale.

Sleep disturbances being considered as a risk factor for headache and the enuretic episode being defined, preliminary data are reported on a clinical epidemiological survey into the greater risk that headache enuresis represents for women despite the fact that the phenomenon is more common among men. Finally it is hypothesised that there is only one qualitative and/or quantitative pathogenetic mechanism that may be transmitter type, or in the light of recent studies, receptorial.

["Precursor phenomena" as a criterion in the choice of prevention of primary headache of the hemicrania type]. / I "fenomeni precursori" come criterio di scelta per la profilassi della cefalea primaria di tipo emicranico.

The importance of headache precursors as expression of latent headache is emphasized. The authors report data from a clinical-epidemiological survey of 322 youngster (143 m and 179 f) aged 4-16 with primary headache aimed at assessing latent time between precursors and onset of headache. The appearance of precursors is considered the time appropriate for prophylaxis.

Discovery of a new gene pool and a high genetic diversity of the chestnut blight fungus Cryphonectria parasitica in Caucasian Georgia.

In this study, we investigated the population genetic structure and possible origins of the plant pathogen Cryphonectria parasitica in Caucasian Georgia, a region within the centre of origin of the host species Castanea sativa. A total of 427 C. parasitica isolates from nine populations were genotyped at 10 microsatellite loci. A high genetic diversity was detected, but the overall Georgian population was dominated by three haplotypes which were present in most individual populations. Two of them have not been previously found in Europe. Bayesian clustering analysis and principal component analysis could not identify their source population, neither in Asia nor in North America. On the other hand, one haplotype is frequent in Central Europe and probably naturally invaded Caucasian Georgia from neighbouring Turkey. Seventy-three haplotypes were unique to specific populations, and 66 of them were represented by a single isolate. Allele patterns suggest that most of these haplotypes emerged locally through sexual recombination between haplotypes of the Georgian and the central European gene pool. Due to the high incidence of haplotypes not otherwise present in Europe, Caucasian Georgia represents an additional source of diversity for the European C. parasitica population.

Population dynamics of the sudden oak death pathogen Phytophthora ramorum in Oregon from 2001 to 2004.

Phytophthora ramorum (Oomycetes) is an emerging plant pathogen in forests in southwestern Oregon (Curry County). Moreover, since 2003 it has been repeatedly isolated from plants in Oregon nurseries. In this study, we analysed the genetic diversity of the P. ramorum population in Oregon from 2001 to 2004 by using microsatellites. A total of 323 isolates (272 from the infested forest; 51 from nurseries) were screened at 10 loci. The overall P. ramorum population in Oregon is characterized by low genetic diversity and has all the hallmarks of an introduced organism. All isolates within the A2 mating type belonged to the same clonal lineage and no recombinant genotypes were found. The forest population (24 genotypes) was dominated by a single multilocus genotype which persisted over years, indicating that eradication efforts in the forest have not completely eliminated inoculum sources. In contrast, genotypic evidence suggests that eradication was effective in nurseries. In 2003 and 2004, a total of 11 genotypes were found in the nurseries (one belonged to the European lineage of P. ramorum) but no genotype was recovered in both sampling years. Significant differentiation and low gene flow were detected between nursery and forest populations. Only two nursery genotypes were also found in the forest, and then at low frequency. Thus, the nursery infestation is not caused by the genotypes observed in Curry County, but likely resulted through introduction of novel genotypes from nurseries out-of-state. This highlights the continued importance of sanitation and quarantine in nurseries to prevent further introduction and spread of P. ramorum.