COMPUTER-AIDED CIVIL AND INFRASTRUCTURE ENGINEERING, Issue 3 2003
Adel Sadek
Existing transportation planning modeling tools have critical limitations with respect to assessing the benefits of intelligent transportation systems (ITS) deployment. In this article, we present a novel framework for developing modeling tools for quantifying ITS deployments benefits. This approach is based on using case,based reasoning (CBR), an artificial intelligence paradigm, to capture and organize the insights gained from running a dynamic traffic assignment (DTA) model. To demonstrate the feasibility of the approach, the study develops a prototype system for evaluating the benefits of diverting traffic away from incident locations using variable message signs. A real,world network from the Hartford area in Connecticut is used in developing the system. The performance of the prototype is evaluated by comparing its predictions to those obtained using a detailed DTA model. The prototype system is shown to yield solutions comparable to those obtained from the DTA model, thus demonstrating the feasibility of the approach. [source]

Range size, taxon age and hotspots of neoendemism in the California flora

DIVERSITY AND DISTRIBUTIONS, Issue 3 2010
Nathan J. B. Kraft
Abstract Aim, Sustaining biological diversity requires the protection of the ecological, evolutionary and landscape-level processes that generate it. Here, we identify areas of high neoendemism in a global diversity hotspot, the California flora, using range size data and molecular-based estimates of taxon age. Location, California, USA. Methods, We compiled distribution and range size data for all plant taxa endemic to California and internal transcribed spacer (ITS)-based age estimates for 337 putative neoendemics (15% of the endemic flora). This information was combined to identify areas in the state with high proportions of young and restricted-range taxa. We overlaid the distribution of neoendemic hotspots on maps of currently protected lands and also explored correlations between our diversity measures and climate. Results, The central coast of California, the Sierra Nevada and the San Bernardino Range contained endemics with the most restricted distributions on average, while areas in the Desert and Great Basin provinces found within the state were composed of the youngest neoendemics on average. Diversity measures that took age and range size into account shifted the estimate of highest endemic diversity in the state towards the Desert and Great Basin regions relative to simple counts of endemic species richness. Our diversity measures were poorly correlated with climate and topographic heterogeneity. Main conclusions, Substantial portions of California with high levels of plant neoendemism fall outside of protected lands, indicating that additional action will be needed to preserve the geographic areas apparently associated with high rates of plant diversification. The neoendemic flora of the deserts appears particularly young in our analyses, which may reflect the relatively recent origin of desert environments within the state. [source]

Large-scale distribution and activity patterns of an extremely low-light-adapted population of green sulfur bacteria in the Black Sea

ENVIRONMENTAL MICROBIOLOGY, Issue 5 2010
Evelyn Marschall
Summary The Black Sea chemocline represents the largest extant habitat of anoxygenic phototrophic bacteria and harbours a monospecific population of Chlorobium phylotype BS-1. High-sensitivity measurements of underwater irradiance and sulfide revealed that the optical properties of the overlying water column were similar across the Black Sea basin, whereas the vertical profiles of sulfide varied strongly between sampling sites and caused a dome-shaped three-dimensional distribution of the green sulfur bacteria. In the centres of the western and eastern basins the population of BS-1 reached upward to depths of 80 and 95 m, respectively, but were detected only at 145 m depth close to the shelf. Using highly concentrated chemocline samples from the centres of the western and eastern basins, the cells were found to be capable of anoxygenic photosynthesis under in situ light conditions and exhibited a photosynthesis,irradiance curve similar to low-light-adapted laboratory cultures of Chlorobium BS-1. Application of a highly specific RT-qPCR method which targets the internal transcribed spacer (ITS) region of the rrn operon of BS-1 demonstrated that only cells at the central station are physiologically active in contrast to those at the Black Sea periphery. Based on the detection of ITS-DNA sequences in the flocculent surface layer of deep-sea sediments across the Black Sea, the population of BS-1 has occupied the major part of the basin for the last decade. The continued presence of intact but non-growing BS-1 cells at the periphery of the Black Sea indicates that the cells can survive long-distant transport and exhibit unusually low maintenance energy requirements. According to laboratory measurements, Chlorobium BS-1 has a maintenance energy requirement of ,1.6,4.9·10,15 kJ cell,1 day,1 which is the lowest value determined for any bacterial culture so far. Chlorobium BS-1 thus is particularly well adapted to survival under the extreme low-light conditions of the Black Sea, and can be used as a laboratory model to elucidate general cellular mechanisms of long-term starvation survival. Because of its adaptation to extreme low-light marine environments, Chlorobium BS-1 also represents a suitable indicator for palaeoceanography studies of deep photic zone anoxia in ancient oceans. [source]

Ecotype diversity in the marine picoeukaryote Ostreococcus (Chlorophyta, Prasinophyceae)

ENVIRONMENTAL MICROBIOLOGY, Issue 6 2005
Francisco Rodríguez
Summary The importance of the cyanobacteria Prochlorococcus and Synechococcus in marine ecosystems in terms of abundance and primary production can be partially explained by ecotypic differentiation. Despite the dominance of eukaryotes within photosynthetic picoplankton in many areas a similar differentiation has never been evidenced for these organisms. Here we report distinct genetic [rDNA 18S and internal transcribed spacer (ITS) sequencing], karyotypic (pulsed-field gel electrophoresis), phenotypic (pigment composition) and physiological (light-limited growth rates) traits in 12 Ostreococcus strains (Prasinophyceae) isolated from various marine environments and depths, which suggest that the concept of ecotype could also be valid for eukaryotes. Internal transcribed spacer phylogeny grouped together four deep strains isolated between 90 m and 120 m depth from different geographical origins. Three deep strains displayed larger chromosomal bands, different chromosome hybridization patterns, and an additional chlorophyll (chl) c -like pigment. Furthermore, growth rates of deep strains show severe photo-inhibition at high light intensities, while surface strains do not grow at the lowest light intensities. These features strongly suggest distinct adaptation to environmental conditions encountered at surface and the bottom of the oceanic euphotic zone, reminiscent of that described in prokaryotes. [source]

Application de la variabilité génétique de l'ADNr chez Monilinia laxa, Monilinia fructigena et Monilinia fructicolaà l'identification des espèces par PCR,

EPPO BULLETIN, Issue 3-4 2000
R. Ioos
Monilinia laxa, Monilinia fructigena et Monilinia fructicola sont des agents de pourriture de fruit et de chancre sur rameau des arbres fruitiers. La région des ITS (internal transcribed spacer) située entre les gènes codant pour les sous-unités ribosomiques 18S et 28S de quatre isolats de M. fructigena et quatre isolats de M. fructigena collectés en France a été amplifiée par PCR et séquencée. L'alignement multiple de ces séquences d'ITS et leur comparaison avec les séquences d'ITS de Monilinia spp. publiées a révélé une très faible variabilité intraspécifique. Par contre, un faible polymorphisme interspécifique a été localisé au niveau de deux régions, respectivement dans l'ITS1 et l'ITS2. Ces deux régions ont été utilisées pour définir des couples d'oligonucléotides espèce-spécifiques. Ces couples d'amorces ont permis d'amplifier par PCR un fragment de 356 pb pour chacune des trois espèces. La spécificité des trois couples d'amorces a été vérifiée avec succès sur une collection de 17 isolats de M. laxa, 18 isolats de M. fructigena et 6 isolats de M. fructicola isolés de différents hôtes. En utilisant des conditions stringentes lors de la PCR, aucune réaction croisée n'a été observée avec les isolats testés. La spécificité du test a été d'autre part vérifiée avec l'ADN total extrait de différentes espèces de champignons, soit phylogénétiquement proche du genre Monilinia soit fréquemment isolées de fruits malades. L'utilisation de cette technique permet d'identifier avec fiabilité un isolaat indéterminé en une seule amplification, en le testant simultanément avec chacun des trois couples d'amorces. De plus, la détection et l'identification des espèces de Monilinia peuvent être réalisés directement à partir de fruits présentant des symptômes. Cette méthode simple et rapide pourrait être particulièrement utile pour détecter M. fructicola qui est un organisme de quarantaine pour l'Europe. [source]

The use of ITS DNA sequence analysis and MALDI-TOF mass spectrometry in diagnosing an infection with Fusarium proliferatum

EXPERIMENTAL DERMATOLOGY, Issue 11 2008
Florian Seyfarth
Abstract:, Although mycoses are among the most common diseases worldwide, infections with Fusarium spp. occur only rarely. Mostly patients suffering from underlying immune deficiency are infected with this mould, resulting in a considerably decreasing prognosis. In immunocompromised patients, cutaneous manifestations are more often associated with Fusarium sp. than with Candida sp. or Aspergillus sp. We describe one patient with acute lymphoblastic leukaemia, who was first treated with chemotherapy after GMALL protocol 07/03. After relapse, the patient was successfully transplanted in second remission with a human leukocyte antigen (HLA)-matched unrelated peripheral blood stem cell graft. Ten months later, the patient died from respiratory insufficiency and recurrence of leukaemia. Previously, Aspergillus antigen was detected in blood. In the latter course, disseminated papules appeared. One of these was examined histologically and mycologically. Conventional cultural diagnostics led to the diagnosis of a fusariosis, further supported by internal transcribed spacer (ITS) sequencing and matrix assisted laser desorption/ionisation,time-of-flight mass spectrometry (MALDI-TOF) mass spectrometry, both determining the isolated strain as Fusarium proliferatum, which is a very infrequent pathogen within this genus. Our investigations underline the potential of MALDI-TOF MS based identification of Fusarium species as an innovative, time and cost efficient alternative to ITS sequencing. [source]

Phylogenetic diversity of Synechococcus strains isolated from the East China Sea and the East Sea

FEMS MICROBIOLOGY ECOLOGY, Issue 3 2009
Dong Han Choi
Abstract Phylogenetic relationships among 33 Synechococcus strains isolated from the East China Sea (ECS) and the East Sea (ES) were studied based on 16S rRNA gene sequences and 16S,23S rRNA gene internal transcribed spacer (ITS) sequences. Pigment patterns of the culture strains were also examined. Based on 16S rRNA gene and ITS sequence phylogenies, the Synechococcus isolates were clustered into 10 clades, among which eight were previously identified and two were novel. Half of the culture strains belonged to clade V or VI. All strains that clustered into novel clades exhibited both phycoerythrobilin and phycourobilin. Interestingly, the pigment compositions of isolates belonging to clades V and VI differed from those reported for other oceanic regions. None of the isolates in clade V showed phycourobilin, whereas strains in clade VI exhibited both phycourobilin and phycoerythrobilin, which is in contrast to previous studies. The presence of novel lineages and the different pigment patterns in the ECS and the ES suggests the possibility that some Synechococcus lineages are distributed only in geographically restricted areas and have evolved in these regions. Therefore, further elucidation of the physiological, ecological, and genetic characteristics of the diverse Synechococcus strains is required to understand their spatial and geographical distribution. [source]

Ericoid mycorrhizal fungi are common root inhabitants of non- Ericaceae plants in a south-eastern Australian sclerophyll forest

FEMS MICROBIOLOGY ECOLOGY, Issue 2 2008
Susan M. Chambers
Abstract Fungi were isolated from the roots of 17 plant species from the families Apiaceae, Cunoniaceae, Cyperaceae, Droseraceae, Fabaceae-Mimosoideae, Lomandraceae, Myrtaceae, Pittosporaceae, Proteaceae and Stylidiaceae at a sclerophyll forest site in New South Wales, Australia. Internal transcribed spacer (ITS) restriction fragment length polymorphism (RFLP) and sequence comparisons indicated that the isolated fungi had affinities to a range of ascomycetes, basidiomycetes and zygomycetes. Four RFLP types had closest affinities to previously identified Helotiales ericoid mycorrhizal (ERM) or Oidiodendron spp. Isolates representing six RFLP types, which were variously isolated from all 17 plant species, formed ERM coils in hair root epidermal cells of Woollsia pungens (Ericaceae) under gnotobiotic conditions. Three of these isolates formed intercellular hyphae, intracellular hyphae and/or microsclerotia, which are typical of dark septate endophyte infection, in roots of Stylidium productum (Stylidiaceae), indicating an ability to form different types of association with roots of different hosts. Overall the data indicate that a broad range of plant taxa may act as repositories for ERM fungi in sclerophyll forest soil. [source]

Retrieval of first genome data for rice cluster I methanogens by a combination of cultivation and molecular techniques

FEMS MICROBIOLOGY ECOLOGY, Issue 2 2005
Christoph Erkel
Abstract We report first insights into a representative genome of rice cluster I (RC-I), a major group of as-yet uncultured methanogens. The starting point of our study was the methanogenic consortium MRE50 that had been stably maintained for 3 years by consecutive transfers to fresh medium and anaerobic incubation at 50 °C. Process-oriented measurements provided evidence for hydrogenotrophic CO2 -reducing methanogenesis. Assessment of the diversity of consortium MRE50 suggested members of the families Thermoanaerobacteriaceae and Clostridiaceae to constitute the major bacterial component, while the archaeal population was represented entirely by RC-I. The RC-I population amounted to more than 50% of total cells, as concluded from fluorescence in situ hybridization using specific probes for either Bacteria or Archaea. The high enrichment status of RC-I prompted construction of a large insert fosmid library from consortium MRE50. Comparative sequence analysis of internal transcribed spacer (ITS) regions revealed that three different RC-I rrn operon variants were present in the fosmid library. Three, approximately 40-kb genomic fragments, each representative for one of the three different rrn operon variants, were recovered and sequenced. Computational analysis of the sequence data resulted in two major findings: (i) consortium MRE50 most likely harbours only a single RC-I genotype, which is characterized by multiple rrn operon copies; (ii) seven genes were identified to possess a strong phylogenetic signal (eIF2a, dnaG, priA, pcrA, gatD, gatE, and a gene encoding a putative RNA-binding protein). Trees exemplarily computed for the deduced amino acid sequences of eIF2a, dnaG, and priA corroborated a specific phylogenetic association of RC-I with the Methanosarcinales. [source]

Potentiality of the cox1 gene in the taxonomic resolution of soil fungi

FEMS MICROBIOLOGY LETTERS, Issue 1 2010
Claire Molitor
Abstract We explored the potential of the cox1 gene in the species resolution of soil fungi and compared it with the nuclear internal transcribed spacer (ITS) and small subunit (SSU)-rDNA. Conserved primers allowing the amplification of the fungal cox1 gene were designed, and a total of 47 isolates of Zygomycota and Ascomycota were investigated. The analysis revealed a lack of introns in >90% of the isolates. Comparison of the species of each of the six studied genera showed high interspecific sequence polymorphisms. Indeed, the average of nucleotide variations (4.2,11%) according to the genus, due mainly to the nucleotide substitutions, led to the taxonomic resolution of all the species studied regarding both ITS and SSU-rDNA, in which <88% were discriminated. The phylogenetic analysis performed after alignment of the cox1 gene across distant fungal species was in accordance with the well-known taxonomic position of the species studied and no overlap was observed between intra- and interspecific variations. These results clearly demonstrated that the cox1 sequences could provide good molecular markers for the determination of the species composition of environmental samples and constitute an important advance to study soil fungal biodiversity. [source]

RESEARCH ARTICLE: Farysizyma gen. nov., an anamorphic genus in the Ustilaginales to accommodate three novel epiphytic basidiomycetous yeast species from America, Europe and Asia

FEMS YEAST RESEARCH, Issue 3 2008
João Inácio
Abstract Among many isolates that resulted from four independent surveys of yeasts associated with plants in Brazil, the USA, Portugal and Taiwan, we have characterized eighteen basidiomycetous strains, two of which were conspecific with the type strain of Rhodotorula acheniorum, whereas the remaining sixteen isolates appeared not to correspond to any previously described species. Microsatellite-PCR fingerprinting with primers M13 and (GTG)5 confirmed that the latter strains formed three genetically distinct groups. Each group was considered to represent a distinct species based on nucleotide sequences of the D1/D2 domains of the 26S rRNA gene and the internal transcribed spacer (ITS) region. Phylogenetic analyses of sequence data placed the putative novel species in a clade with R. acheniorum and the dimorphic smut fungus Farysia chardoniana. A novel anamorphic genus, Farysizyma, is created to accommodate the three undescribed species, which were named Farysizyma itapuensis, Farysizyma setubalensis and Farysizyma taiwaniana. A new combination, Farysizyma acheniorum, is proposed for R. acheniorum, which may represent the yeast-phase anamorph of Farysia thuemenii. [source]

Pseudozyma jejuensis sp. nov., a novel cutinolytic ustilaginomycetous yeast species that is able to degrade plastic waste

FEMS YEAST RESEARCH, Issue 6 2007
Hyuk-Seong Seo
Abstract An ustilaginomycetous anamorphic yeast, isolated from orange leaves on Jeju island in South Korea, represents a novel Pseudozyma species according to morphologic and physiologic findings and molecular taxonomic analysis using the D1/D2 domains of the large subunit (26S) rRNA gene and the internally transcribed spacer (ITS) 1+2 regions. The name Pseudozyma jejuensis sp. nov. is proposed for this novel species, with OL71T (=KCTC 17482T=CBS 10454T) as type strain. In the present study, we have also demonstrated that Pseudozyma jejuensis OL71 is capable of producing cutinase and degrading polycaprolactone. These results suggest that Pseudozyma jejuensis or its cutinase may be useful for the biological degradation of plastic waste. [source]

The teleomorph of Chalara fraxinea, the causal agent of ash dieback

FOREST PATHOLOGY, Issue 5 2009
T. Kowalski
Summary Ash dieback, caused by the pathogen Chalara fraxinea, is an emerging lethal disease of Fraxinus excelsior, threatening the host species in large parts of Europe. The ascomycete Hymenoscyphus albidus (Helotiaceae, Helotiales) was identified as the teleomorph of C. fraxinea by culturing from ascospores, morphological comparison and nuclear ribosomal internal transcribed spacer (ITS) sequencing. [source]

PCR primers for identification of Sirococcus conigenus and S. tsugae, and detection of S. conigenus from symptomatic and asymptomatic red pine shoots

FOREST PATHOLOGY, Issue 3 2008
D. R. Smith
Summary Regions of diversity in the internal transcribed spacer (ITS) sequences of Sirococcus species were exploited to design primer pairs used in a PCR-based method for the identification of the conifer shoot blight pathogen Sirococcus conigenus and the closely related fungus Sirococcus tsugae. The specificity of each primer pair for the respective fungus, detection limits and utility for detection from host material were confirmed. The S. conigenus primers were then used to detect this pathogen in tissues of symptomatic or apparently healthy red pine shoots collected at six locations in Wisconsin and Michigan and results compared with those obtained using a cultural assay. For needles, bark and wood of symptomatic shoots, the mean frequencies of detection of S. conigenus using the PCR-based methods were consistent (,7.5 out of 10) and always greater than for the cultural assay. Detection from symptomatic shoots using the cultural assay was more frequent from needles than from bark or wood. Both the PCR-based method and the cultural assay detected S. conigenus in similar frequencies from asymptomatic shoots, although less frequently than from symptomatic shoots. The efficiency of the PCR-based method and its utility for direct testing of host material should make it particularly useful in areas where multiple shoot blight pathogens are found. [source]

Occurrence of the wattle wilt pathogen, Ceratocystis albifundus on native South African trees

FOREST PATHOLOGY, Issue 5 2007
J. Roux
Summary Ceratocystis albifundus causes the disease known as wattle wilt of non-native Acacia mearnsii trees in South Africa, Uganda and Kenya. Infection results in rapid wilt and death of susceptible trees and stem cankers on more tolerant trees. It has been suggested that C. albifundus is indigenous to southern Africa, possibly having spread from native Protea spp. to non-native A. mearnsii and A. decurrens trees. Although C. albifundus has been collected from Protea spp., these reports are based on limited records for which only aged herbarium specimens exist. During surveys of wound-infecting fungi on native tree species in South Africa, a fungus resembling C. albifundus was collected from Protea gaguedi, Acacia caffra, Burkea africana, Combretum molle, C. zeyheri, Faurea saligna, Ochna pulchra, Ozoroa paniculosa and Terminalia sericea. The identity of the fungus was confirmed as C. albifundus, using comparisons of DNA sequence data for the ITS and 5.8S gene of the rRNA operon. In pathogenicity trials, lesions were produced on C. molle and A. caffra, with some trees beginning to die at the termination of the experiment. This study represents the first report of C. albifundus from native tree species in South Africa and provides unequivocal evidence that the fungus occurs naturally on native Protea spp. The wide host range of C. albifundus, as well as its abundance on these indigenous hosts lends further support to the view that it is a native African pathogen. [source]

Phylogeographic variation among isolates of the Sirococcus conigenus P group

FOREST PATHOLOGY, Issue 1 2007
H. Konrad
Summary In this study the phylogeographic variation among isolates of the Sirococcus conigenus P group and the phylogenetic relationships of S. conigenus with Sirococcus clavigignenti-juglandacearum and other species previously placed in the genus Sirococcus were investigated. A collection of 33 isolates originating from Picea, Pinus and Larix in Europe, North America and Bhutan were characterized by sequence analyses of the internal transcribed spacer (ITS) region (including ITS1, 5.8S ribosomal DNA, ITS2) of the nuclear rDNA and a portion of the , -tubulin gene. In phylogenetic analyses most isolates from pine, spruce and larch formed a distinct clade, representing the P group of S. conigenus, which was separated from the T group of this pathogen. Four isolates from Picea in Europe and Canada formed a third clade within S. conigenus and these isolates are referred to as the S group. The P group consisted of five distinct ITS haplotypes, which partly differed in their optimum growth temperature and their growth rates at 25°C on malt extract agar. Nested clade analysis resolved the five haplotypes into three distinct clades and revealed significant genetic/geographic associations for some of the haplotypes. Parsimony analysis of the small subunit (18S) ribosomal DNA sequences confirmed the phylogenetic affinities between S. conigenus and S. clavigignenti-juglandacearum. In contrast, Godronia cassandrae and Hormococcus conorum, which formerly had been placed in the genus Sirococcus, were found to be only distantly related to S. conigenus and S. clavigignenti-juglandacearum. [source]

Mycosphaerella species associated with leaf disease of Eucalyptus globulus in Ethiopia

FOREST PATHOLOGY, Issue 4 2006
Alemu Gezahgne
Summary Eucalyptus spp. are among the most widely planted exotic trees in Ethiopia. Several damaging leaf pathogens are known from Eucalyptus spp. worldwide. Of these, Mycosphaerella spp. are among the most important, causing the disease known as Mycosphaerella leaf disease (MLD). Characteristic symptoms of MLD include leaf spot, premature defoliation, shoot and twig dieback. Recent disease surveys conducted in Ethiopian Eucalyptus plantations have revealed disease symptoms similar to those caused by Mycosphaerella spp. These symptoms were restricted to E. globulus trees growing in several localities in south, south western and western Ethiopia. The aim of this study was to identify the fungi associated with this disease. This was achieved by examining ascospore germination patterns, anamorph associations and sequence data from the Internal Transcribed Spacer (ITS) region of the rRNA operon, for representative isolates. Several different ascospore germination patterns were observed, suggesting that more than one species of Mycosphaerella is responsible for MLD on E. globulus in Ethiopia. Analysis of sequence data showed that three Mycosphaerella spp., M. marksii, M. nubilosa and M. parva were present. This is the first report of these three species from Ethiopia and represents a valuable basis on which to build further studies in the region. Résumé Les Eucalyptus comptent parmi les essences d'arbres exotiques les plus plantées en Ethiopie. Plusieurs pathogènes foliaires sont connus dans le monde pour occasionner des dégâts sur Eucalyptus. Parmi ceux-ci, les espèces de Mycosphaerella sont parmi les plus importantes, causant la maladie connue comme Maladie Foliaire àMycosphaerella (MFM, MLD en anglais). Les symptômes caractéristiques de la MFM comprennent des taches foliaires, une défoliation précoce et des dépérissements de pousses et de rameaux. Des campagnes de surveillance menées récemment dans les plantations éthiopiennes d'Eucalyptus ont révélé la présence de tels symptômes. Ces symptômes sont uniquement observés sur E. globulus dans plusieurs localités du sud, sud-ouest et ouest de l'Ethiopie. L'objectif de cette étude était d'identifier les champignons associés à cette maladie. Pour cela, des isolats représentatifs ont étéétudiés pour les modalités de germination des ascospores, les anamorphes associés ainsi que les données de séquence de la région ITS de l'opéron ADNr. Différentes modalités de germination des ascospores ont été observées, suggérant que plusieurs espèces de Mycosphaerella seraient associées à la MFM sur E. globulus en Ethiopie. L'analyse des données de séquence a montré la présence de 3 espèces : M. marksii, M. nubilosa et M. parva. Ceci constitue la première mention de ces 3 espèces en Ethiopie et une première étape pour envisager d'autres études dans cette région. Zusammenfassung Eucalyptus -Arten sind die am häufigsten angepflanzten exotischen Bäume in Äthiopien. An Eucalyptus kommen verschiedene Blattkrankheiten vor, wobei die Mycosphaerella -Arten als Verursacher der Mycosphaerella -Blattkrankheit (MLD) am bedeutendsten sind. Charakteristische Symtpome der MLD sind Blattnekrosen und vorzeitiger Blattfall sowie Trieb- und Zweigsterben. Bei der Inventur von Krankheiten in äthiopischen Eucalyptusplantagen wurden Symptome entdeckt, die denen von Mycosphaerella spp. ähnlich waren. Diese traten nur an E. globulus lokal in S-, SW- und W-Äthiopien auf. Ziel dieser Untersuchung war es, die damit assoziierten Pilze zu identifizieren. Hierzu wurde an repräsentativen Isolaten das Keimverhalten der Ascosporen, das Vorkommen von Anamorphen und die ITS-Sequenz des rRNA-Operons untersucht. Es wurden verschiedene Keimungstypen der Ascosporen beobachtet, was darauf schliessen liess, dass mehr als eine Mycosphaerella -Art für die Krankheit an E. globulus in Äthiopien verantwortlich ist. Anhand der Sequenzen wurden M. marksii, M. nubilosa und M. parva identifiziert. Dies ist der Erstnachweis für diese drei Arten in Äthiopien und eine Grundlage für weitere Studien. [source]

Studies on anastomosis groups of Rhizoctonia solani isolates causing disease in two forest nurseries in Poland

FOREST PATHOLOGY, Issue 2 2006
S. St, pniewska-Jarosz
Summary Thirty-eight isolates of Rhizoctonia spp. were isolated from Scots pine (Pinus sylvestris) seedlings with damping-off symptoms, originating from two forest nurseries in central-west Poland (Wronczyn and Jarocin) and from diseased seedlings grown in soil from Wronczyn nursery. Majority of these isolates (79%) had multinucleate cells and were identified as Rhizoctonia solani. The remaining isolates were recognized as binucleate Rhizoctonia spp. R. solani isolates were characterized using hyphal anastomosis and were divided into five anastomosis groups (AG). The most prevalent was AG5 (37% of isolates), followed by AG2-1 (30%) and 27% of the isolates were identified as AG4. Groups AG1-IB and AG2-2 were only represented by single isolates. The virulence recorded as mortality (in percentage) was comparatively high for binucleate and multinucleate isolates of Rhizoctonia spp. Sequence analysis of the polymerase chain reaction (PCR)-amplified internal transcribed spacer (ITS) rDNA region was used for phylogenetic analysis. The dendrogram showed that isolates were distinctly separated based on their AG types and there was no relationship between pathogenicity on Scots pine seedlings and the AG to which the isolates belong to. The results are discussed with respect to pathogenic potential of the various AG groups. Résumé Trente-huit isolats de Rhizoctonia spp. ont été isolés de semis de Pin sylvestre (Pinus sylvestris) présentant des symptômes de fonte, dans deux pépinières forestières du Centre-Ouest de la Pologne (Wronczyn and Jarocin) et de semis malades élevés dans du sol provenant de la pépinière de Wronczyn. La majorité de ces isolats (79%) ont des cellules multi-nucléées et ont été identifiés comme des Rhizoctonia solani. Le reste des isolats ont été reconnus comme des Rhizoctonia spp. binucléés. Les isolats de R. solani ont été caractérisés en utilisant l'anastomose d'hyphes et répartis dans cinq groupes d'anastomoses (AG). Le plus important est le groupe AG5 (37% des isolats), suivi par AG2-1 (30%) et AG4 (27%). Les groupes AG1-IB et AG2-2 sont représentés chacun par seulement un isolat. La virulence, estimée par le pourcentage de mortalité, est relativement forte pour les isolats binucléés et multinucléés de Rhizoctonia spp. L'analyse des séquences de la région ITS de l'ADNr amplifiées par PCR a été utilisée pour l'analyse phylogénétique. Le dendrogramme montre que les isolats sont séparés selon leur groupe d'anastomose mais il n'y a pas de relation entre le groupe d'anastomose et la virulence sur semis de Pin sylvestre. Les résultats sont discutés dans la perspective du pouvoir pathogène des différents groupes d'anastomoses. Zusammenfassung Von Kiefernsämlingen (Pinus sylvestris) mit Umfallkrankheit, die aus zwei Forstbaumschulen in Zentral-Westpolen stammten (Wronczyn und Jarocin) und aus erkrankten Sämlingen, die in Bodenproben aus der Baumschule Wronczyn kultiviert worden waren, wurden 38 Stämme von Rhizoctonia spp. isoliert. Die meisten dieser Isolate (79%) hatten vielkernige Zellen und wurden als R. solani identifiziert. Die restlichen Isolate waren zweikernige Rhizoctonia spp. Die Isolate von R. solani wurden durch Anastomosierungstests charakterisiert und fünf Anastomosierungsgruppen zugeordnet. Die häufigste Gruppe war AG5 (37% der Isolate), gefolgt von AG2-1 (30%) und AG4 (27%). Die Gruppe AG1-IB und AG2-2 waren nur durch einzelne Isolate vertreten. Die Virulenz (gemessen als % Mortalität) war sowohl für zweikernige als auch für vielkernige Isolate vergleichsweise hoch. Mit den Sequenzen der PCR-amplifizierten ITS-rDNA-Region wurde eine phylogenetische Analyse durchgeführt. Das Dendrogramm zeigte, dass die Isolate aufgrund ihrer Zugehörigkeit zu den Anastomosierungsgruppen deutlich voneinander getrennt waren, und es bestand keine Beziehung zwischen ihrer Virulenz gegenüber Kiefernsämlingen und der Gruppenzugehörigkeit. Die Befunde werden im Hinblick auf das pathogene Potential der verschiedenen Anastomosierungsgruppen diskutiert. [source]

Rhizoctonia solani AG 2-1 as a causative agent of cotyledon rot on European beech (Fagus sylvatica)

FOREST PATHOLOGY, Issue 6 2005
A. M. Hietala
Summary Rhizoctonia solani was frequently isolated in the Italian Alps from nursery-grown European beech (Fagus sylvatica) seedlings displaying symptoms of cotyledon rot. Koch's postulates were verified and mode of infection of the associated isolates was investigated with light and scanning electron microscopy. Population structure of the pathogen was investigated by scoring the anastomosis reaction type in pairings between different isolates from the same seedbed. One pathogen genotype showed a large distribution area within the seedbed, this implying that the inoculum had been building up in the seedbed over a longer time period. Hyphal anastomosis tests and sequence analysis of the internal transcribed spacer (ITS) region of ribosomal DNA indicated that the pathogen belongs to AG 2-1 of R. solani. ITS sequence analysis indicates that the isolates from beech are closely related to R. solani isolates causing a disease on tulip. The striking similarities between the two diseases are discussed. Résumé Rhizoctonia solani a fréquemment été isolé de semis de hêtre (Fagus sylvatica) présentant des symptômes de pourriture des cotylédons dans une pépinière forestière des Alpes italiennes. Les postulats de Koch ont été vérifiés et le mode d'infection étudié par microscopie optique et électronique à balayage. La structure de la population de l'agent pathogène a étéétudiée en examinant les réactions d'anastomoses dans les confrontations par paires des isolats d'un même lit de semences. Un génotype particulier s'est avéré largement distribué dans le lit de semence, suggérant soit une accumulation de l'inoculum pendant une longue période soit que ce génotype est capable de reproduction homocaryotique, favorisant sa dispersion. Les tests d'anastomose et l'analyse de la séquence de la région ITS de l'ADN ribosomal indiquent que l'agent pathogène appartient au groupe AG 2-1 de R. solani. L'analyse de la séquence de l'ITS montre que les isolats de hêtre sont proches d'isolats de R. solani pathogènes sur tulipe. Les ressemblances frappantes entre les deux maladies et la gestion de la maladie sur hêtre sont discutées. Zusammenfassung In einer Forstbaumschule in den italienischen Alpen wurde Rhizoctonia solani häufig aus Buchenkeimlingen (Fagus sylvatica) mit Symptomen einer Kotyledonenfäule isoliert. Die Koch'schen Postulate wurden erfüllt und die Art der Infektion der beteiligten Isolate wurde licht- und rasterelektronen-mikroskopisch untersucht. Die Populationsstruktur des Pathogens wurde anhand der Reaktionstypen (Anastomosierungsverhalten) in Paarungsversuchen mit den unterschiedlichen Isolaten aus demselben Saatbeet untersucht. Ein Kompatibilitätstyp war innerhalb des Saatbeetes weit verbreitet, was darauf hindeutet, dass sich das Inokulum über einen längeren Zeitraum dort angereichert hatte und/oder der Genotyp homokaryotisch fruchtet, was seine Ausbreitung fördert. Die Anastomosierungstests und die ITS-Sequenzanalyse der ribosomalen DNA ergaben, dass der Erreger zu der AG 2-1 Gruppe R. solani gehört. Die ITS-Sequenzen deuten darauf hin, dass die Isolate von Buche mit den R. solani, Isolaten verwandt sind, die an Tulpen pathogen sind. Die auffallende Ähnlichkeit der beiden Krankheiten und das Management der Erkrankung an Buche wird diskutiert. [source]

Identification of Lophodermium seditiosum and L. pinastri in Swedish forest nurseries using species-specific PCR primers from the ribosomal ITS region

FOREST PATHOLOGY, Issue 3 2005
E. Stenström
Summary Lophodermium seditiosum is a serious needle pathogen on pine, particularly in nurseries, and there is a need to detect the pathogen during its latent phase. The internal transcribed spacer (ITS) regions of the rDNA of L. seditiosum and L. pinastri were amplified with universal primers and sequenced. Sequence comparisons of the two species allowed the design of species-specific primers for the ITS regions. The primers were between 18 and 24 bp long with a minimum of 3 bp differences between the species. These primer pairs did not give any amplification of DNA from any other of the examined fungal species or from healthy Pinus sylvestris needles. It was also possible to identify either L. seditiosum or L. pinastri in infected needles with and without signs of infection using these primer pairs. The method was found to be very useful for detection of latent infections of L. seditiosum in P. sylvestris needles in nurseries. Résumé Lophodermium seditiosum est un pathogène important des aiguilles sur pins, particulièrement en pépinières, et il serait nécessaire de détecter le pathogène dans sa phase latente. Les régions ITS de L. seditiosum et L. pinastri ont été amplifiées avec des amorces universelles et séquencées. La comparaison de la séquence des deux espèces a permis de développer des amorces spécifiques pour chaque espèce dans la région ITS. Les amorces ont une longueur de 18 à 24 paires de bases avec un minimum de 3 paires de bases de différence entre espèces. Ces amorces n'ont produit aucune amplification avec l'ADN des autres espèces de champignons testées ou les aiguilles saines de Pinus sylvestris. Il a également été possible de détecter L. seditiosum ou L. pinastri avec ces amorces dans des aiguilles infectées avec ou sans signe d'infection. Cette méthode s'avère très utile pour la détection d'infections latentes de L. seditiosum dans les aiguilles de P. sylvestris en pépinières. Zusammenfassung Lophodermium seditiosum ist ein starkes Nadelpathogen an Kiefern, speziell in Baumschulen. Für den Einsatz von Bekämpfungsmassnahmen wäre es von Vorteil, wenn man das Pathogen bereits während der Latenzperiode nachweisen könnte. Die ITS Regionen der ribosomalen DNA von L. seditiosum und L. pinastri wurden mit Standardprimern amplifiziert und sequenziert. Vergleiche der Sequenzen der beiden Arten erlaubten die Entwicklung von artspezifischen Primern für die ITS Regionen. Die Primerpaare waren zwischen 18 and 24 Basenpaaren lang und wiesen einen Unterschied von mindestens drei Nukleotiden auf. Die DNA von allen anderen untersuchten Pilzarten und von gesunden Pinus sylvestris Nadeln liessen sich mit keinem dieser Primerpaare amplifiziern. Lophodermium seditiosum und L. pinastri konnten mit den Primerpaaren in infizierten Nadeln mit und ohne Symptome direkt nachgewiesen werden. Die Methode eignete sich vorzüglich zum Nachweis von latenten Infektionen von L. seditiosum in P. sylvestris Nadeln in Baumschulen. [source]

Development of species-specific PCR primers on rDNA for the identification of European Armillaria species

FOREST PATHOLOGY, Issue 5 2003
G. Sicoli
Summary Attempts to design species-specific PCR primers from six European Armillaria species in the ribosomal RNA genes are reported. Primers were developed on the basis of the nucleotide sequence variability of the internal transcribed spacers (ITS) and the intergenic spacer (IGS1) of the ribosomal DNA. Four sets of primers gave specific PCR products for Armillaria tabescens, Armillaria mellea and Armillaria ostoyae. However, due to the high sequence similarities between Armillaria borealis and Armillaria ostoyae and between Armillaria cepistipes and Armillaria gallica no species specific amplification was obtained for these taxa. Résumé Des essais ont été réalisés pour obtenir des amorces PCR spécifiques de 6 espèces européennes d'Armillaria dans les gènes de l'ARNr. Les amorces ont été développées sur la base de la variabilité de séquence nucléotidique dans les ITS et IGS (IGS1) de l'ADN ribosomal. Quatre couples d'amorces ont permis d'obtenir des produits PCR spécifiques pour A. tabescens, A. mellea et A. ostoyae. Cependant, compte tenu des très fortes similarités de séquence entre A. borealis et A. ostoyae, et entre A. cepistipes et A. gallica, il n'a pas été obtenu d'amplification spécifique pour ces taxons. Zusammenfassung Es wird über Versuche berichtet, artspezifische Primer für sechs europäische Armillariaarten in der Region der ribosomalen RNA-Gene zu entwickeln. Als Grundlage dafür diente die Variabilität der Nukleotidsequenzen der ITS- und der IGS 1-Region der ribosomalen DNA. Vier Primerpaare ergaben spezifische PCR-Produkte für A. tabescens, A. mellea und A. ostoyae. Dagegen wurden aufgrund der grossen Ähnlichkeit der Sequenzen von A. borealis und A. ostoyae sowie von A. cepistipes und A. gallica für diese Taxa keine artspezifischen Amplifikationsprodukte erhalten. [source]

Molecular diagnosis of Phytophthora lateralis in trees, water, and foliage baits using multiplex polymerase chain reaction

FOREST PATHOLOGY, Issue 5 2001
L. M. Winton
A polymerase chain reaction (PCR)-based protocol for detection of Phytophthora lateralis in plant tissues and water is described. Base-pair (bp) deletions in both of the ribosomal DNA internal transcribed spacer (ITS) regions in P. lateralis were used to design complementary PCR primer sequences that amplify a 738 bp fragment only if P. lateralis DNA is present in the sample. Universal control primers based on conserved sequences of the nuclear ribosomal small subunit are included in a multiplexed reaction, providing an internal check on the procedure. The universal primers amplify an approximately 550 bp fragment that is common to plants, protists, and true fungi. The procedure reliably detects P. lateralis in cedar stem tissues and in roots. Positive reactions were obtained with as few as 200 P. lateralis zoospores in water. Diagnostic moléculaire par PCR multiplex pour détecter Phytophthora lateralis dans les arbres, l'eau et le feuillage utilisé comme piège Un protocole basé sur la PCR est décrit pour détecter Phytophthora lateralis dans les tissus végétaux et l'eau. Des délétions de paires de bases dans chacune des régions ITS de l'ADN ribosomal de P. lateralis ont été utilisées pour définir des amorces de PCR qui n'amplifient un fragment de 738 paires de bases que si l'ADN de P. lateralis est présent dans l'échantillon. Des amorces universelles basées sur des régions conservées de la petite sous-unité de l'ADN ribosomal nucléaire ont été incluses dans une réaction de PCR multiplex, fournissant ainsi un témoin interne de la réaction. Ces amorces universelles amplifient un fragment de 550 pb qui est commun aux plantes, aux protistes et aux champignons vrais. Ce protocole permet la détection de P. lateralis dans les tiges et dans les racines du Chamaecyparis. Des réactions positives ont été obtenues avec seulement 200 zoospores de P. lateralis dans l'eau. Molekulare Diagnose von Phytophthora lateralis in Bäumen, Wasser und als Köder benutzten Blättern mittels Multiplex-PCR Eine auf der PCR beruhende Methode zum Nachweis von Phytophthora lateralis in Pflanzengeweben und Wasser wird beschrieben. Deletionen in den beiden ITS Regionen der ribosomalen DNA von P. lateralis wurden zur Synthese von PCR-Primern ausgenutzt, die ein 738 Basenpaare langes Fragment nur dann amplifizieren, wenn P. lateralis in der Probe vorhanden ist. Universelle Primer, die konservierten Sequenzen der kleinen Unterheit der ribosomalen Kern-DNA entsprechen, wurden als interne Kontrollen in die Multiplex-PCR miteinbezogen. Diese Primer amplifizieren ein ungefähr 550 Basenpaare langes Fragment, das sowohl bei Pflanzen als auch bei Protisten und höheren Pilzen vorkommt. Mit der Methode liess sich P. lateralis im Stamm und in den Wurzeln von Lawsons Scheinzypresse verlässlich nachweisen. Für den Nachweis von P. lateralis im Wasser waren mindestens 200 Zoosporen nötig. [source]

Rapid and accurate identification of microorganisms contaminating cosmetic products based on DNA sequence homology

INTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 6 2005
Y. Fujita
Synopsis The aim of this study was to develop rapid and accurate procedures to identify microorganisms contaminating cosmetic products, based on the identity of the nucleotide sequences of the internal transcribed spacer (ITS) region of the ribosomal RNA coding DNA (rDNA). Five types of microorganisms were isolated from the inner portion of lotion bottle caps, skin care lotions, and cleansing gels. The rDNA ITS region of microorganisms was amplified through the use of colony-direct PCR or ordinal PCR using DNA extracts as templates. The nucleotide sequences of the amplified DNA were determined and subjected to homology search of a publicly available DNA database. Thereby, we obtained DNA sequences possessing high similarity with the query sequences from the databases of all the five organisms analyzed. The traditional identification procedure requires expert skills, and a time period of approximately 1 month to identify the microorganisms. On the contrary, 3,7 days were sufficient to complete all the procedures employed in the current method, including isolation and cultivation of organisms, DNA sequencing, and the database homology search. Moreover, it was possible to develop the skills necessary to perform the molecular techniques required for the identification procedures within 1 week. Consequently, the current method is useful for rapid and accurate identification of microorganisms, contaminating cosmetics. Résumé Le but de cette étude est de développer une procédure rapide et fiable pour identifier les micro-organismes contaminant les produits cosmétiques. Cette procédure repose sur l'identification des séquences des nucléotides des espaceurs transcrits internes (Internal Transcribed Spacer ou région ITS), de l'ADN codant pour l'ARN ribosomique (rADN). Cinq types de micro-organismes sont isolés sur la partie intérieure des bouchons des flacons de lotions pour le soin de la peau et de gels lavants. Les régions ITS rADN des micro-organismes sont amplifiées grâce à l'utilisation de la méthode ,colony-direct PCR, ou ,ordinal PCR, en utilisant les extraits d'ADN comme matrices. Les séquences de nucléotides de l'ADN amplifiées sont évaluées et soumises à une recherche homologique dans une librairie d'ADN disponible au public. Ainsi, grâce aux bases de données, nous obtenons des séquences d'ADN qui possèdent une similaritéélevée avec les séquences recherchées des cinq organismes analysés. La procédure d'identification classique exige des compétences d'experts et une période d'environ un mois pour identifier les micro-organismes. D'autre part, il faut 3 à 7 jours pour terminer toutes les procédures utilisées dans la méthode ici décrite, y compris l'isolation et la culture des organismes, le séquençage de l'ADN et la recherche dermatologique dans les bases de données. De plus, il est possible en 1 semaine de développer les compétences nécessaires pour mettre en ,uvre les techniques moléculaires requises pour les procédures d'identification. Cette méthode est donc utile pour une identification rapide et fiable des micro-organismes qui contaminent les cosmétiques. [source]

Parallel Algorithms for Dynamic Shortest Path Problems

INTERNATIONAL TRANSACTIONS IN OPERATIONAL RESEARCH, Issue 3 2002
Ismail Chabini
The development of intelligent transportation systems (ITS) and the resulting need for the solution of a variety of dynamic traffic network models and management problems require faster-than-real-time computation of shortest path problems in dynamic networks. Recently, a sequential algorithm was developed to compute shortest paths in discrete time dynamic networks from all nodes and all departure times to one destination node. The algorithm is known as algorithm DOT and has an optimal worst-case running-time complexity. This implies that no algorithm with a better worst-case computational complexity can be discovered. Consequently, in order to derive algorithms to solve all-to-one shortest path problems in dynamic networks, one would need to explore avenues other than the design of sequential solution algorithms only. The use of commercially-available high-performance computing platforms to develop parallel implementations of sequential algorithms is an example of such avenue. This paper reports on the design, implementation, and computational testing of parallel dynamic shortest path algorithms. We develop two shared-memory and two message-passing dynamic shortest path algorithm implementations, which are derived from algorithm DOT using the following parallelization strategies: decomposition by destination and decomposition by transportation network topology. The algorithms are coded using two types of parallel computing environments: a message-passing environment based on the parallel virtual machine (PVM) library and a multi-threading environment based on the SUN Microsystems Multi-Threads (MT) library. We also develop a time-based parallel version of algorithm DOT for the case of minimum time paths in FIFO networks, and a theoretical parallelization of algorithm DOT on an ,ideal' theoretical parallel machine. Performances of the implementations are analyzed and evaluated using large transportation networks, and two types of parallel computing platforms: a distributed network of Unix workstations and a SUN shared-memory machine containing eight processors. Satisfactory speed-ups in the running time of sequential algorithms are achieved, in particular for shared-memory machines. Numerical results indicate that shared-memory computers constitute the most appropriate type of parallel computing platforms for the computation of dynamic shortest paths for real-time ITS applications. [source]

Estimating time dependent O-D trip tables during peak periods

JOURNAL OF ADVANCED TRANSPORTATION, Issue 3 2000
Srinivas S. Pulugurtha
Intelligent transportation systems (ITS) have been used to alleviate congestion problems arising due to demand during peak periods. The success of ITS strategies relies heavily on two factors: 1) the ability to accurately estimate the temporal and spatial distribution of travel demand on the transportation network during peak periods, and, 2) providing real-time route guidance to users. This paper addresses the first factor. A model to estimate time dependent origin-destination (O-D) trip tables in urban areas during peak periods is proposed. The daily peak travel period is divided into several time slices to facilitate simulation and modeling. In urban areas, a majority of the trips during peak periods are work trips. For illustration purposes, only peak period work trips are considered in this paper. The proposed methodology is based on the arrival pattern of trips at a traffic analysis zone (TAZ) and the distribution of their travel times. The travel time matrix for the peak period, the O-D trip table for the peak period, and the number of trips expected to arrive at each TAZ at different work start times are inputs to the model. The model outputs are O-D trip tables for each time slice in the peak period. 1995 data for the Las Vegas metropolitan area are considered for testing and validating the model, and its application. The model is reasonably robust, but some lack of precision was observed. This is due to two possible reasons: 1) rounding-off, and, 2) low ratio of total number of trips to total number of O-D pair combinations. Hence, an attempt is made to study the effect of increasing this ratio on error estimates. The ratio is increased by multiplying each O-D pair trip element with a scaling factor. Better estimates were obtained. Computational issues involved with the simulation and modeling process are discussed. [source]

Differential identification of Bacillus anthracis from environmental Bacillus species using microarray analysis

JOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2006
J.E. Burton
Abstract Aims:, To determine whether microarray analysis could be employed for the differential identification of a range of environmental Bacillus sp. from four strains of Bacillus anthracis. Methods and Results:, Oligonucleotide probes were designed that were specific to virulence factor genes of B. anthracis (pag, lef and cap), the variable number tandem repeat region of the B. anthracis vrrA gene and to the 16S-23S rRNA intergenic transcribed spacer region (ITS) and pleiotropic regulator (plcR) regions of the Bacillus cereus subgroup species. Generic probes were also designed to hybridize with conserved regions of the 16S rRNA genes of Bacillus (as a positive control), Neisseria sp., Pseudomonas sp., Streptococcus sp., Mycobacterium sp. and to all members of the Enterobacteriaceae to allow simultaneous detection of these bacteria. Identification of B. anthracis was found to rely entirely on hybridization of DNA specific to regions of the pag, lef and cap genes. Cross-reaction was observed between B. anthracis and other Bacillus species with all the other Bacillus probes tested. Results obtained using microarray hybridizations were confirmed using conventional microbiological techniques and found to have very high comparability. Conclusions:, Microarray-based assays are an effective method for the identification of B. anthracis from mixed-culture environmental samples without problems of false-positivity that have been observed with conventional PCR assays. Significance and Impact of the Study:, Identification of environmental Bacillus sp. by conventional PCR is prone to potential for reporting false-positives. This study provides a method for the exclusion of such isolates. [source]

Effect of wine yeast monoculture practice on the biodiversity of non- Saccharomyces yeasts

JOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2004
M.A. Ganga
Abstract Aims:, The objective of this work was to study the effect of the use of Saccharomyces cerevisiae monocultures over the biodiversity of non- Saccharomyces yeasts in wine-producing areas in Chile. Methods and Results:, Microvinifications were carried out with grape musts of two areas. In one of them, the fermentation is carried out mainly in a spontaneous manner, whereas in the other the musts are inoculated with commercial yeasts. The isolated yeasts were identified by the internal transcribed (ITS)/restriction fragment length polymorphism technique. In the industrial production area less variability of yeast genera was observed as compared with the traditional area, an observation that is greatest at the end of the fermentation. Furthermore, a study of the production of extracellular enzymes was done. The majority of the yeasts showed at least one of the activities assayed with the exception of , -glycosidase. Conclusion:, The results suggest that in the industrialized area the diversity of yeasts is less in the traditional area. Likewise, the potentiality of the non- Saccharomyces yeasts as enzyme producers with industrial interest has been confirmed. Significance and Impact of the Study:, This study shows the negative effect of the use of monocultures over the biodiversity of yeasts in wine-producing regions. [source]

Surviving climate changes: high genetic diversity and transoceanic gene flow in two arctic,alpine lichens, Flavocetraria cucullata and F. nivalis (Parmeliaceae, Ascomycota)

JOURNAL OF BIOGEOGRAPHY, Issue 8 2010
József Geml
Abstract Aim, We examined genetic structure and long-distance gene flow in two lichenized ascomycetes, Flavocetraria cucullata and Flavocetraria nivalis, which are widespread in arctic and alpine tundra. Location, Circumpolar North. Methods, DNA sequences were obtained for 90 specimens (49 for F. cucullata and 41 for F. nivalis) collected from various locations in Europe, Asia and North America. Sequences of the nuclear internal transcribed spacer (ITS) + 5.8S ribosomal subunit gene region were generated for 89 samples, and supplemented by beta-tubulin (BTUB) and translation elongation factor 1-alpha gene (EF1) sequences for a subset of F. cucullata specimens. Phylogenetic, nonparametric permutation methods and coalescent analyses were used to assess population divergence and to estimate the extent and direction of migration among continents. Results, Both F. cucullata and F. nivalis were monophyletic, supporting their morphology-based delimitation, and had high and moderately high intraspecific genetic diversity, respectively. Clades within each species contained specimens from both North America and Eurasia. We found only weak genetic differentiation among North American and Eurasian populations, and evidence for moderate to high transoceanic gene flow. Main conclusions, Our results suggest that both F. cucullata and F. nivalis have been able to migrate over large distances in response to climatic fluctuations. The high genetic diversity observed in the Arctic indicates long-term survival at high latitudes, whereas the estimated migration rates and weak geographic population structure suggest a continuing long-distance gene flow between continents that has prevented pronounced genetic differentiation. The mode of long-distance dispersal is unknown, but wind dispersal of conidia and/or ascospores is probably important in the open arctic landscapes. The high genetic diversity and efficient long-distance dispersal capability of F. cucullata and F. nivalis suggest that these species, and perhaps other arctic lichens as well, will be able to track their potential niche in the changing Arctic. [source]

Evolution and biogeography of the austral genus Phyllocladus (Podocarpaceae)

JOURNAL OF BIOGEOGRAPHY, Issue 10 2004
Steven J. Wagstaff
Abstract Aim, To infer evolutionary relationships within the genus Phyllocladus and among its close relatives by phylogenetic analysis of DNA sequences. Interpret the inferred relationships in association with the fossil record to examine the origin and diversification of the genus. Location, Australasia. Methods, Phylogenetic analyses of rbcL, matK and internal transcribed spacer (ITS) sequences representing all of the extant species of Phyllocladus and a selection of outgroups from Podocarpaceae and Araucariaceae. Results, The rbcL and matK sequences exhibit little variation within Phyllocladus, but ally its members to Podocarpaceae although its immediate sister remains unclear. The ITS sequences resolve all five species of Phyllocladus and two intraspecific ecotypes of P. alpinus. Main conclusions,Phyllocladus forms a distinct lineage that diverged early in the evolutionary history of Podocarpaceae. The fossil record indicates that the genus was more widely distributed and morphologically diverse during the early Tertiary than at present. Although of Mesozoic origin, the level of sequence variation within Phyllocladus suggests that the extant species radiated during the late Tertiary c. 6.3 ± 0.9 Ma. New Zealand is the present centre of species diversity. [source]

Elevated genetic heterogeneity and Pleistocene climatic instability: inferences from nrDNA in New Zealand Coprosma (Rubiaceae)

JOURNAL OF BIOGEOGRAPHY, Issue 7 2002
Stephen R. Wichman
Aim To examine patterns of hybridization and genotype mixing within the genus Coprosma J.R.Forst. & G.Forst. (Rubiaceae). Location New Zealand Methods Nucleotide sequence was determined for the internal transcribed spacer (ITS) and external transcribed spacer (ETS) regions of nuclear ribosomal DNA for fifty individuals from thirty-six taxa within the New Zealand component of the genus Coprosma. Results Mixed sequences were found to be widespread in Coprosma. Direct sequencing of ITS polymerase chain reaction (PCR) products from seven polyploid taxa showed evidence of sequence mixtures. Cloning and sequencing of individual PCR products from two polyploids confirmed the presence of multiple templates, one of which corresponded to that of a diploid. Intra-individual heterogeneity was also seen in a hybrid diploid taxon, with the mixed nucleotides corresponding to those of the parental lineages. Finally the ITS sequences of twenty-two diploid taxa showed that eleven contained intra-individual heterogeneity. Conclusions We conclude that the widespread occurrence of sequence mixtures in Coprosma results from of frequent hybridization. We also conclude that concerted evolution of the ITS and ETS regions is depressed. We propose that these characteristics evolved as a mechanism to maintain high levels of heterogeneity and suggest that this is adaptive for Coprosma in climatically unstable and physically complex New Zealand landscapes. These landscapes have been subjected to repeated oscillations between stadial and interstadial environments during the Pleistocene. [source]