Evidence for homoploid speciation in Phytophthora alni supports taxonomic reclassification in this species complex.

Alder decline has been a problem along European watercourses since the early 1990s. Hybridization was identified as the main cause of this emerging disease. Indeed, the causal agent, a soil-borne pathogen named Phytophthora alni subsp. alni (Paa) is the result of interspecific hybridization between two taxa, Phytophthora alni subsp. multiformis (Pam) and Phytophthora alni subsp. uniformis (Pau), initially identified as subspecies of Paa. The aim of this work was to characterize the ploidy level within the P. alni complex that is presently poorly understood. For that, we used two complementary approaches for a set of 31 isolates of Paa, Pam and Pau: (i) quantification of allele copy number of three single-copy nuclear genes using allele-specific real-time PCR and (ii) comparison of the genome size estimated by flow cytometry. Relative quantification of alleles of the three single-copy genes showed that the copy number of a given allele in Paa was systematically half that of its parents Pau or Pam. Moreover, DNA content estimated by flow cytometry in Paa was equal to half the sum of those in Pam and Pau. Our results therefore suggest that the hybrid Paa is an allotriploid species, containing half of the genome of each of its parents Pam and Pau, which in turn are considered to be allotetraploid and diploid, respectively. Paa thus results from a homoploid speciation process. Based on published data and on results from this study, a new formal taxonomic name is proposed for the three taxa Paa, Pam and Pau which are raised to species status and renamed P. ×alni, P. ×multiformis and P. uniformis, respectively.

Biogeographical patterns and determinants of invasion by forest pathogens in Europe.

A large database of invasive forest pathogens (IFPs) was developed to investigate the patterns and determinants of invasion in Europe. Detailed taxonomic and biological information on the invasive species was combined with country-specific data on land use, climate, and the time since invasion to identify the determinants of invasiveness, and to differentiate the class of environments which share territorial and climate features associated with a susceptibility to invasion. IFPs increased exponentially in the last four decades. Until 1919, IFPs already present moved across Europe. Then, new IFPs were introduced mainly from North America, and recently from Asia. Hybrid pathogens also appeared. Countries with a wider range of environments, higher human impact or international trade hosted more IFPs. Rainfall influenced the diffusion rates. Environmental conditions of the new and original ranges and systematic and ecological attributes affected invasiveness. Further spread of established IFPs is expected in countries that have experienced commercial isolation in the recent past. Densely populated countries with high environmental diversity may be the weakest links in attempts to prevent new arrivals. Tight coordination of actions against new arrivals is needed. Eradication seems impossible, and prevention seems the only reliable measure, although this will be difficult in the face of global mobility.

Is the emergence of Dothistroma needle blight of pine in France caused by the cryptic species Dothistroma pini?

Dothistroma needle blight (DNB) emerged in France in the past 15 years. This disease is induced by two closely related species: Dothistroma septosporum and D. pini. Although both species are currently present in France, only D. septosporum was reported in the past. We investigated whether a recent arrival of D. pini in France could be a cause of the DNB emergence. We analyzed herbarium specimens of pine needles with DNB symptoms using polymerase chain reaction techniques to study the past frequency of D. pini in France. We also determined the present distribution within the country of D. septosporum and D. pini and compared it with the spatial pattern of DNB reported in the Département de la Santé des Forêts (DSF; French forest health monitoring agency) database. Although D. pini was detected on herbarium specimens from 1907 and 1965, it was not frequent in France in the past. Today, it is frequent, although not present throughout the country, being absent from the north and the east. There is no relationship between the D. pini distribution in France and the spatial pattern of DNB reported in the DSF database. Thus, the emergence of DNB in France cannot be explained by a recent arrival of D. pini.

Effect of poplar genotypes on mycorrhizal infection and secreted enzyme activities in mycorrhizal and non-mycorrhizal roots.

The impact of ectomycorrhiza formation on the secretion of exoenzymes by the host plant and the symbiont is unknown. Thirty-eight F(1) individuals from an interspecific Populus deltoides (Bartr.)×Populus trichocarpa (Torr. & A. Gray) controlled cross were inoculated with the ectomycorrhizal fungus Laccaria bicolor. The colonization of poplar roots by L. bicolor dramatically modified their ability to secrete enzymes involved in organic matter breakdown or organic phosphorus mobilization, such as N-acetylglucosaminidase, ß-glucuronidase, cellobiohydrolase, ß-glucosidase, ß-xylosidase, laccase, and acid phosphatase. The expression of genes coding for laccase, N-acetylglucosaminidase, and acid phosphatase was studied in mycorrhizal and non-mycorrhizal root tips. Depending on the genes, their expression was regulated upon symbiosis development. Moreover, it appears that poplar laccases or phosphatases contribute poorly to ectomycorrhiza metabolic activity. Enzymes secreted by poplar roots were added to or substituted by enzymes secreted by L. bicolor. The enzymatic activities expressed in mycorrhizal roots differed significantly between the two parents, while it did not differ in non-mycorrhizal roots. Significant differences were found between poplar genotypes for all enzymatic activities measured on ectomycorrhizas except for laccases activity. In contrast, no significant differences were found between poplar genotypes for enzymatic activities of non-mycorrhizal root tips except for acid phosphatase activity. The level of enzymes secreted by the ectomycorrhizal root tips is under the genetic control of the host. Moreover, poplar heterosis was expressed through the enzymatic activities of the fungal partner.

Are plant pathogen populations adapted for encounter with their host? A case study of phenological synchrony between oak and an obligate fungal parasite along an altitudinal gradient.

Biotrophic fungal pathogens are expected to have adapted to their host plants for phenological synchrony, to optimize the possibility of contacts leading to infections. We investigated the patterns and causes of variation in phenological synchrony in the oak-powdery mildew pathosystem, a major disease in natural ecosystems. The study was carried out along an altitudinal gradient, representing a wide temperature range, in mature oak stands. Both sporulation (pathogen infective stage) and oak flushing (host susceptible stage) were delayed with increasing elevation, but with a significantly different sensitivity for the two species. This resulted in a variable host-pathogen synchrony along the gradient. A common garden experiment did not give evidence of among-population genetic differentiation (past adaptation) for fungal phenology. This could be explained by the high phenotypic variation in phenology within host populations, precluding selection on fungal phenology at the population scale, but possibly favouring adaptation at the within-population scale. Phenotypic plasticity was the major cause of the observed variation in the phenology of the fungal populations.

Tracking the invasion: dispersal of Hymenoscyphus fraxineus airborne inoculum at different scales.

Ash dieback is caused by an invasive pathogen Hymenoscyphus fraxineus, which emerged in Europe in the 1990s and jeopardizes the management of ash stands. Although the biological cycle of the pathogen is well understood, its dispersal patterns via airborne spores remain poorly described. We investigated the seasonal and spatial patterns of dispersal in France using both a passive spore-trapping method coupled with a real-time PCR assay and reports of ash dieback based on symptom observations. Spores detection varies from year to year with a detection ability of 30-47% depending on meteorological conditions, which affect both production of inoculum and efficiency of the trapping. Nevertheless, our results are consistent and we showed that the sporulation peak occurred from June to August and that spores were detected up to 50-100 km ahead of the disease front, proving the presence of the pathogen before any observation of symptoms. The spore dispersal gradient was steep, most of inoculum remaining within 50 m of infected ashes. Two dispersal kernels were fitted using Bayesian methods to estimate the mean dispersal distance of H. fraxineus from inoculum sources. The estimated mean distances of dispersal, either local or regional scale, were 1.4 km and 2.6 km, respectively, the best fitting kernel being the inverse power-law. This information may help to design disease management strategies.

Risk factors for the phytophthora-induced decline of alder in northeastern france.

ABSTRACT A lethal disease of common alder caused by Phytophthora alni, a new hybrid pathogen, has been spreading in Europe since the early 1990s. In 2004, we conducted an epidemiological survey in northeastern France to determine disease frequency and to investigate the impact of environmental factors on disease prevalence. Seventy-eight plots in the Rhin-Meuse basin were investigated. The survey was structured to enable critical examination of the possible impact of nitrogen pollution of the river water on disease prevalence. P. alni-induced alder decline was common throughout northeastern France. Altogether, disease was found in 80% of the plots containing alder, with 16% of all the alders affected. Striking differences existed between watercourse types. Lower proportions of diseased alders were found in watercourse types with rapid water flow, such as mountain streams of the Vosges and piedmont or watercourses on steep calcareous slopes, than in the slow watercourses of the low-lying valleys of the calcareous plateaus and of the clayey plains. Disease prevalence was not related to the total oxidized nitrogen concentration of the water. However, prevalence increased with the mean summer temperature of the river water and where clayey soils were found in the river banks. The results of this work can be used for the assessment of P. alni-induced alder decline risks in affected European countries and in areas where the disease could be introduced.

First Report of Phytophthora ramorum on Ornamental Plants in France.

In April 2002, Phytophthora ramorum was associated with twig blight and brown spots on Rhododendron spp. leaves from a nursery in France. The isolate was identified by its morphological characters on V8 agar: slow growth, deciduous and semipapillate sporangia, and abundant production of large chlamydospores (3). The identification was confirmed by ITS rDNA sequencing. During 2002, P. ramorum was also isolated from diseased Viburnum tinus and V. × bodnantense plants exhibiting symptoms of wilting and stem base discoloration. Subsequently, repeated surveys for P. ramorum were carried out in nurseries and areas surrounding nurseries throughout France. Since 2004, a large range of known hosts were investigated in approximately 2,000 nurseries and 200 other sites each year. P. ramorum was detected exclusively in nurseries at 29 locations in 2002, 9 in 2003, 23 in 2004, 17 in 2005, and 19 in 2006. Rhododendron spp. and occasionally V. tinus were the major hosts. In addition, the pathogen was detected for the first time on Pieris japonica in two nurseries in 2005 and on Camellia sp. in one nursery in 2006 from plants exhibiting leaf and twig blight. In both cases, P. ramorum had already been detected on Rhododendron spp. in the same nurseries. Most of the infected plants were found in northwestern France (Bretagne and Pays-de-la-Loire), or came from this region, which is the main rhododendron-growing area in France. In some cases, plants were imported from Belgium or the Netherlands. P. ramorum was also detected in a nursery in soil close to diseased Rhododendron spp. plants and pond water used for irrigation by using a combination of baiting with Rhododendron spp. leaves and PCR assay with species-specific primers (1). Overall, approximately 1% of the investigated nurseries were found positive each year, and this ratio was quite stable from 2004 to 2006. To date, P. ramorum has not been detected outside of nurseries, although many surveys were conducted on the west coast of France where the risk is considered to be high because of a favorable mild and humid climate and the presence of suitable hosts. In addition, 78 isolates of P. ramorum collected between 2002 and 2004 on Rhododendron spp. and V. tinus were found to be of A1 mating type based on pairings with P. cryptogea A1 and A2 mating types (2). References: (1) K. J. Hayden et al. Phytopathology 94:1075, 2004. (2) S. Werres and B. Zielke J. Plant Dis. Prot. 110:129, 2003. (3) S. Werres et al. Mycol. Res. 105:1155, 2001.

Construction and characterization of a partial library of yeast artificial chromosomes from human chromosome 21.

We report a protocol for cloning large DNA fragments in yeast artificial chromosomes (YAC). A partial library has been constructed from a somatic hybrid containing chromosome 21 as the single source of human DNA. About 4.0 Mb of human DNA was recovered in 17 YAC clones. Three clones were analyzed by in situ hybridization and mapped on chromosome 21. One clone hybridized with the chromosome 21 centromeric region and may provide new insight both on the molecular structure of centromere and on the localization of Alzheimer disease gene.

[Study of alpha-satellite DNA in cosmid libraries, specific for chromosomes 13, 21, and 22, using fluorescence in situ hybridization]. / Issledovanie al'fa-satellitnykh DNK v sostave kosmidnykh bibliotek, spetsifichnykh dlia khromosome 13, 21, i 22, s pomoshch'iu fliuorestsentnoi gibridizatsii in situ.

Fluorescent in situ hybridization (FISH) was employed in mapping the alpha-satellite DNA that was revealed in the cosmid libraries specific for human chromosomes 13, 21, and 22. In total, 131 clones were revealed. They contained various elements of centromeric alphoid DNA sequences of acrocentric chromosomes, including those located close to SINEs, LINEs, and classical satellite sequences. The heterochromatin of acrocentric chromosomes was shown to contain two different groups of alphoid sequences: (1) those immediately adjacent to the centromeric regions (alpha 13-1, alpha 21-1, and alpha 22-1 loci) and (2) those located in the short arm of acrocentric chromosomes (alpha 13-2, alpha 21-2, and alpha 22-2 loci). Alphoid DNA sequences from the alpha 13-2, alpha 21-2, and alpha 22-2 loci are apparently not involved in the formation of centromeres and are absent from mitotically stable marker chromosomes with a deleted short arm. Robertsonian translocations t(13q; 21q) and t(14q; 22q), and chromosome 21p-. The heterochromatic regions of chromosomes 13, 21, and 22 were also shown to contain relatively chromosome-specific repetitive sequences of various alphoid DNA families, whose numerous copies occur in other chromosomes. Pools of centromeric alphoid cosmids can be of use in further studies of the structural and functional properties of heterochromatic DNA and the identification of centromeric sequences. Moreover, these clones can be employed in high-resolution mapping and in sequencing the heterochromatic regions of the human genome. The detailed FISH analysis of numerous alphoid cosmid clones allowed the identification of several new, highly specific DNA probes of molecular cytogenetic studies--in particular, the interphase and metaphase analyses of chromosomes 2, 9, 11, 14, 15, 16, 18, 20, 21-13, 22-14, and X.

A rapid and simple method to isolate and characterize highly polymorphic markers from the centromeric regions of the human chromosomes.

Using oligonucleotide primers complementary to the 3' ends of either the Alu or the L1Hs consensus sequences in conjunction with a primer complementary to alpha satellite subsets specific to different human chromosomes, it was possible to detect and characterize polymorphisms originating from the microsatellites which are often present downstream these repetitive elements. The methodology does not require cloning, sequencing or synthesis of specific primers. Centromeric location was confirmed by linkage analysis, in situ hybridization and sequencing. The method is proposed for the generation of polymorphic markers from all centromeric regions.

TaqI reveals two independent alphoid polymorphisms on human chromosomes 13 and 21.

We have analysed the TaqI patterns obtained with an alphoid DNA probe specific for human chromosomes 13 and 21 in a number of unrelated individuals, as well as in the somatic hybrid WA 17 which carries chromosome 21 as a unique human chromosome. In certain individuals, two types of extra bands are superimposed over the relatively simple basic banding pattern exhibited by all individuals. Thus, three independent allele-specific DNA patterns are defined. The basic and normal organization of the alpha satellite in chromosome 21 consists of tandemly arranged arrays of repeats representing 11 times the 171-bp monomer of the alphoid DNA sequences. The supernumerary bands found in some individuals are organized in tandemly arranged subsets of repeats representing 18 times and 9.5 times the 171bp basic monomer, respectively. These less fragment alleles segregate in a Mendelian fashion. Linkage analyses suggest that they originate from chromosomes 13 and 21, respectively.

Analysis of pericentromeric chromosome 21 specific YAC clones by FISH: identification of new markers for molecular-cytogenetic application.

Fluorescence in situ hybridization (FISH) of chromosome 21 specific yeast artificial chromosome (YAC) clones after Alu-PCR (polymerase chain reaction) amplification has been used to find new region-specific DNA probes for the heterochromatic region of chromosome 21. Six overlapping YAC clones from a pericentromeric contig map (region 21cen-21q11) were analyzed. Four YAC clones were characterized as hybridizing to several chromosomal locations. They are, therefore, either chimeric or shared by different chromosomes. Two of them containing alphoid satellite DNA, are localized at the centromeric regions of chromosomes 13 and 21 (clone 243A11), and on 13cen, 21cen and 1q3 (clone 781G5); the two others are localized at both 21q11 and 13q2 (clone 759D3), and at 18p (clone 770B3). Two YACs were strongly specific for chromosome 21q11 only (clones 124A7 and 881D2). These YACs were used effectively as probes for identifications of chromosome 21 during metaphase and interphase analysis of 12 individuals, including three families with Down syndrome offspring, and 6 aminocyte samples. The location of YAC clones on 21q11 close to the centromeric region allows the application of these clones as molecular probes for the analysis of marker chromosomes with partial deletions of the long arm as well as for pre- and postnatal diagnosis of trisomy 21 when alphoid or more distal region-specific DNA probes are uninformative. Overlapping YAC clones covering human chromosome 21q may be systematically used to detect a set of band-specific DNA probes for molecular-cytogenetic application.

Structural organization and polymorphism of the alpha satellite DNA sequences of chromosomes 13 and 21 as revealed by pulse field gel electrophoresis.

More than 30 unrelated individuals were analysed by pulse field gel electrophoresis for the alphoid centromeric sequences of chromosomes 13 and 21. These individuals had DNA patterns all different from each other and were most probably heterozygous at both loci. When several nuclear families were analysed in this manner, segregation was shown to be Mendelian, and no recombination event was detected over the 150 meioses scored in this study. Alphoid DNA sequences, therefore, constitute highly polymorphic centromeric markers, which can be used in linkage analysis for loci close to the centromeres of chromosomes 13 and 21.

On the mode of evolution of alpha satellite DNA in human populations.

The hypothesis that highly reiterated satellite DNAs in present-day populations evolve by molecular mechanisms that create, by saltatory amplification steps, new long arrays of satellite DNA, and that such long arrays are used for homogenization purposes, has been tested both in mouse and in humans. In mouse, the data obtained are consistent with this hypothesis. This was tested in more detail on chromosomes 13 and 21 of the human genome. A Centre d'Etudes du Polymorphisme Humain family, which in some individuals exhibits strong supplementary DNA bands following TaqI restriction endonuclease digestion and conventional gel electrophoresis, was analyzed by pulse field gel electrophoresis following restriction by BamHI. The supplementary bands on chromosome 13 (18 times the basic alpha satellite DNA repeat) and on chromosome 21 (a 9.5-mer) segregated with centromeric alpha satellite DNA blocks of 5 and 5.3 megabases, respectively. These are by far the largest alpha satellite block lengths seen in all chromosome 13 and chromosome 21 centromeric sequences so far analyzed in this manner. The possibility that these supplementary alpha satellite sequences were created in single individuals by saltatory amplification steps is discussed in light of our own data and that published by others. It is proposed that deletion events and unequal cross-overs, which both occur in large satellite DNA arrays, contribute to the homogenization of size and sequence of the alpha satellite DNA on most chromosomes of humans.

Organization of the variant domains of alpha satellite DNA on human chromosome 21.

The de novo creation of long, homogeneous, satellite DNA domains was postulated previously to occur by saltatory amplification. In this paper, pulsed field gel electrophoresis analysis of the alpha satellite DNA block organization of the human chromosome 21 supports this hypothesis. Double-dimension electrophoresis indicated that the variant copies of the basic alpha satellite repeat of chromosome 21 are organized in a single 3,150 Kb-long domain. It was also established that the other satellite DNAs found in man (beta, II, and III) are organized independently of the alpha satellite DNA block of the same chromosome.

Discrimination between alpha-satellite DNA sequences from chromosomes 21 and 13 by using polymerase chain reaction.

alpha-Satellite subfamilies from chromosomes 21 and 13 are almost identical in sequence and cannot be distinguished from each other by hybridization techniques. A general method based on membrane-bound PCR is described here, allowing the discrimination of alpha-satellite DNA sequences from each of these two chromosomes, after detection by Southern blot hybridization. The PCR conditions were developed using somatic hybrid DNAs. The method was tested in membrane-bound PCR by using the alpha-satellite bands from a Southern blot of a CEPH family. The chromosomal origin of these bands, previously determined by linkage analysis, was confirmed by this method.

High resolution multicolor fluorescence in situ hybridization using cyanine and fluorescein dyes: rapid chromosome identification by directly fluorescently labeled alphoid DNA probes.

We tested DNA probes directly labeled by fluorescently labeled nucleotides (Cy3-dCTP, Cy5-dCTP, FluorX-dCTP) for high resolution uni- and multicolor detection of human chromosomes and analysis of centromeric DNA organization by in situ hybridization. Alpha-satellite DNA probes specific to chromosomes 1, 2, 3, 4 + 9, 5 + 19, 6, 7, 8, 10, 11, 13 + 21, 14 + 22, 15, 16, 17, 18, 20, 22, X and Y were suitable for the accurate identification of human chromosomes in metaphase and interphase cells. Cy3-labeled probes had several advantages: (1) a high level of fluorescence (5-10 times more compared with fluorescein-labeled probes); (2) a low level of fluorescence in solution, allowing the detection of target chromosomes in situ during hybridization without the washing of slides; and (3) high resistance to photobleaching during prolonged (1-2 h) exposure to strong light, thus allowing the use of a high energy mercury lamp or a long integration time during image acquisition in digital imaging microscopy for the determination of weak signals. For di- and multicolor fluorescence in situ hybridization (FISH), we successfully used different combinations of directly fluorophorated probes with preservation of images by conventional microscopy or by digital imaging microscopy. FluorX and Cy3 dyes allowed the use of cosmid probes for mapping in a one-step hybridization experiment. Cyanine-labeled fluorophorated DNA probes offer additional possibilities for rapid chromosome detection during a simple 15-min FISH procedure, and can be recommended for basic research and clinical studies, utilizing FISH.