A new TCAD simulation method for direct CMOS electron detectors optimization.

A custom CMOS image sensor hardened by design is characterized in a transmission electron microscope, with the aim to extract basic parameters such as the quantum efficiency, the modulation transfer function and finally the detective quantum efficiency. In parallel, a new methodology based on the combination of Monte Carlo simulation of electron distributions and TCAD simulations is proposed and performed on the same detector, and for the first time the basic parameters of a direct CMOS electron detector are extracted thanks to the TCAD. The methodology is validated by means of the comparison between experimental and simulation results. This simulation method may be used for the development of future electron detectors.

A Permutation-Randomization Approach to Test the Spatial Distribution of Plant Diseases.

The analysis of the spatial distribution of plant diseases requires the availability of trustworthy geostatistical methods. The mean distance tests (MDT) are here proposed as a series of permutation and randomization tests to assess the spatial distribution of plant diseases when the variable of phytopathological interest is categorical. A user-friendly software to perform the tests is provided. Estimates of power and type I error, obtained with Monte Carlo simulations, showed the reliability of the MDT (power > 0.80; type I error < 0.05). A biological validation on the spatial distribution of spores of two fungal pathogens causing root rot on conifers was successfully performed by verifying the consistency between the MDT responses and previously published data. An application of the MDT was carried out to analyze the relation between the plantation density and the distribution of the infection of Gnomoniopsis castanea, an emerging fungal pathogen causing nut rot on sweet chestnut. Trees carrying nuts infected by the pathogen were randomly distributed in areas with different plantation densities, suggesting that the distribution of G. castanea was not related to the plantation density. The MDT could be used to analyze the spatial distribution of plant diseases both in agricultural and natural ecosystems.

Population genetic analyses provide insights on the introduction pathway and spread patterns of the North American forest pathogen Heterobasidion irregulare in Italy.

A population genetics approach is used to identify the most likely introduction site and introduction pathway for the North American forest pathogen Heterobasidion irregulare using 101 isolates from six sites in Italy and 34 isolates from five sites in North America. Diversity indices based on sequences from ten loci indicate the highest diversity in Italy is found in Castelfusano/Castelporziano and that diversity progressively decreases with increasing distance from that site. AMOVA, Bayesian clustering and principal coordinates analyses based on 12 SSR loci indicate high levels of gene flow among sites, high frequency of admixing, and fail to identify groups of genotypes exclusive to single locations. Cumulatively, these analyses suggest the current infestation is the result of multiple genotypes expanding their range from a single site. Based on two sequenced loci, a single source site in North America could provide enough variability to explain the variability observed in Italy. These results support the notion that H. irregulare was introduced originally in Castelporziano: because Castelporziano has been sealed off from the rest of the world for centuries except for a camp set up by the US military in 1944, we conclude the fungus may have been transported in infected wood used by the military. Finally, spatial autocorrelation analyses using SSR data indicate a significant under-dispersion of alleles up to 0.5-10 km, while a significant overdispersion of alleles was detected at distances over 80 km: these ranges can be used to make predictions on the likely dispersal potential of the invasive pathogen.

Vulnerability of CMOS image sensors in Megajoule Class Laser harsh environment.

CMOS image sensors (CIS) are promising candidates as part of optical imagers for the plasma diagnostics devoted to the study of fusion by inertial confinement. However, the harsh radiative environment of Megajoule Class Lasers threatens the performances of these optical sensors. In this paper, the vulnerability of CIS to the transient and mixed pulsed radiation environment associated with such facilities is investigated during an experiment at the OMEGA facility at the Laboratory for Laser Energetics (LLE), Rochester, NY, USA. The transient and permanent effects of the 14 MeV neutron pulse on CIS are presented. The behavior of the tested CIS shows that active pixel sensors (APS) exhibit a better hardness to this harsh environment than a CCD. A first order extrapolation of the reported results to the higher level of radiation expected for Megajoule Class Laser facilities (Laser Megajoule in France or National Ignition Facility in the USA) shows that temporarily saturated pixels due to transient neutron-induced single event effects will be the major issue for the development of radiation-tolerant plasma diagnostic instruments whereas the permanent degradation of the CIS related to displacement damage or total ionizing dose effects could be reduced by applying well known mitigation techniques.

Amplified fragment length polymorphism and sequence analyses reveal massive gene introgression from the European fungal pathogen Heterobasidion annosum into its introduced congener H. irregulare.

The paucity of fungal species known to be currently hybridizing has significantly hindered our understanding of the mechanisms driving gene introgression in these eukaryotic microbes. Here, we describe an area of hybridization and gene introgression between the invasive plant pathogen Heterobasidion irregulare (introduced from North America) and the native H. annosum in Italy. A STRUCTURE analysis of amplified fragment length polymorphism data for 267 individuals identified gene introgression in 8-42% of genotypes in the invasion area, depending on site. Data indicate that introgression is mostly occurring unilaterally from the native to the invasive species and is responsible for 5-45% of genomes in admixed individuals. Sequence analysis of 11 randomly selected and unlinked loci for 30 individuals identified introgression at every locus, thus confirming interspecific gene flow involves a large number of loci. In 37 cases, we documented movement of entire alleles between the two species, but in 7 cases, we also documented the creation of new alleles through intralocus recombination. Sequence analysis did not identify enrichment of either transcriptionally different nonsynonymous alleles or of transcriptionally identical synonymous alleles. These findings may suggest introgression is occurring randomly for extant alleles without an obvious enrichment process driven by selection. However, further studies are needed to ensure selection is not at work elsewhere in the genome.

Optimization of sampling procedures for DNA-based diagnosis of wood decay fungi in standing trees.

AIMS: To develop fast and reliable sampling procedures for DNA-based diagnosis of wood decay fungi in standing trees. METHODS AND RESULTS: A total of 250 trees were tested for the presence of a suite of wood decay fungi by collecting wood frass obtained by drilling each tree once with a 4-mm-diameter, 43-cm-long bit. We identified at least one of 11 target wood decay fungi in 56 trees through multiplex PCR assays. The presence of target wood decay taxa was further investigated in these 56 trees, by analysing independently wood from each of six drillings. Results were then compared with those obtained using sampling schemes differing in terms of number and position of drillings. Samples of 1-4 drillings were either analysed separately, and the results were combined, or pooled together before analysis was performed. In comparison with taxa identified by the analysis of six drillings, diagnostic efficiency ranged from 56.6% for the scheme based on a single drill to 96.8% for the scheme based on four drillings analysed separately. Both schemes significantly differ (P < 0.05) from those based on two and three drillings, whose efficiency was 72.6% and 83.9%, respectively. Diagnostic efficiency of pooled samples was comparable to that of samples analysed separately. CONCLUSIONS: Highest diagnostic efficiency was obtained by analysing wood from four drillings. It is advisable to pool samples deriving from different drillings to reduce laboratory costs. SIGNIFICANCE AND IMPACT OF THE STUDY: Fast and reliable sampling procedures make DNA-based diagnosis more suitable for tree inspection procedures.

Inferences on the phylogeography of the fungal pathogen Heterobasidion annosum, including evidence of interspecific horizontal genetic transfer and of human-mediated, long-range dispersal.

Fungi in the basidiomycete species complex Heterobasidion annosum are significant root-rot pathogens of conifers throughout the northern hemisphere. We utilize a multilocus phylogenetic approach to examine hypotheses regarding the evolution and divergence of two Heterobasidion taxa associated with pines: the Eurasian H. annosum sensu stricto and the North American H. annosum P intersterility group (ISG). Using DNA sequence information from portions of two nuclear and two mitochondrial loci, we infer phylogenetic relationships via parsimony, Bayesian and median-joining network analysis. Analysis of isolates representative of the entire known geographic range of the two taxa results in monophyletic sister Eurasian and North American lineages, with North America further subdivided into eastern and western clades. Genetically anomalous isolates from the Italian presidential estate of Castelporziano are always part of a North American clade and group with eastern North America, upholding the hypothesis of recent, anthropogenically mediated dispersal. P ISG isolates from Mexico have phylogenetic affinity with both eastern and western North America. Results for an insertion in the mitochondrial rDNA suggest this molecule was obtained from the Heterobasidion S ISG, a taxon sympatric with the P ISG in western North America. These data are compatible with an eastern Eurasian origin of the species, followed by dispersal of two sister taxa into western Eurasia and into eastern North America over a Beringean land bridge, a pattern echoed in the phylogeography of other conifer-associated basidiomycetes.

A multiplex PCR-based method for the detection and early identification of wood rotting fungi in standing trees.

AIMS: The goal of this research was the development of a PCR-based assay to identify important decay fungi from wood of hardwood tree species in northern temperate regions. METHODS AND RESULTS: Eleven taxon-specific primers were designed for PCR amplification of either nuclear or mitochondrial ribosomal DNA regions of Armillaria spp., Ganoderma spp., Hericium spp., Hypoxylon thouarsianum var. thouarsianum, Inonotus/Phellinus-group, Laetiporus spp., Perenniporia fraxinea, Pleurotus spp., Schizophyllum spp., Stereum spp. and Trametes spp. Multiplex PCR reactions were developed and optimized to detect fungal DNA and identify each taxon with a sensitivity of at least 1 pg of target DNA in the template. This assay correctly identified the agents of decay in 82% of tested wood samples. CONCLUSIONS: The development and optimization of multiplex PCRs allowed for reliable identification of wood rotting fungi directly from wood. SIGNIFICANCE AND IMPACT OF THE STUDY: Early detection of wood decay fungi is crucial for assessment of tree stability in urban landscapes. Furthermore, this method may prove useful for prediction of the severity and the evolution of decay in standing trees.

Invasion of European pine stands by a North American forest pathogen and its hybridization with a native interfertile taxon.

It was recently reported that North American (NA) individuals of the forest pathogen Heterobasidion annosum were found in a single pine stand near Rome, in association with the movement of US troops during World War II. Here, we report on some aspects of the invasion biology of this pathogen in Italian coastal pinewoods, and on its interaction with native (EU) Heterobasidion populations. Spores of Heterobasidion were sampled using woody traps in pine stands along 280 km of coast around Rome. DNA of single-spore colonies was characterized by two sets of nuclear and one set of mitochondrial taxon-specific polymerase chain reaction primers. NA spores were found not only in a single site, but in many locations over a wide geographic area. Invasion occurred at an estimated rate of 1.3 km/year through invasion corridors provided by single trees, and not necessarily by sizable patches of forests. Within the 100-km long range of expansion, the NA taxon was dominant in all pure pine stands. Because abundance of the EU taxon is low and identical among stands within and outside the area invaded by NA individuals, we infer that the exotic population has invaded habitats mostly unoccupied by the native species. Discrepancy between a mitochondrial and a nuclear marker occurred in 3.8% of spores from one site, a mixed oak-pine forest where both taxa were equally represented. Combined phylogenetic analyses on nuclear and mitochondrial loci confirmed these isolates were recombinant. The finding of hybrids indicates that genetic interaction between NA and EU Heterobasidion taxa is occurring as a result of their current sympatry.

First Report of Pestalotiopsis funerea on Cupressocyparis leylandii in Italy.

Pestalotiopsis funerea (Desm.) Steyaert occurs worldwide and is reported as an opportunistic invader of conifers. It can also be have as a primary pathogen. During the spring of 2001, leaf yellowing was observed on mature and young Cupressocyparis leylandii trees in some nurseries in northern Italy. Symptoms were first observed on twig tips and progressed toward the stem during the summer, resulting in twig dieback and death of branches up to 15 mm. Dark acervuli were present in the cankers. Isolations were made from cankers and necrotic twigs by removing the outer tissue and placing wood slivers from affected tissues on potato dextrose agar. P. funerea was isolated from 88 and 72% of cankers and necrotic twigs, respectively. Four isolates were selected for inoculation tests. Inoculations were made in September on the upper part of stems of 4-year-old pot-grown C. leylandii saplings. The outer layer of the bark (5 × 3 mm) was removed, and a 5-mm-diameter mycelial plug on malt extract agar (MEA) was placed on the wound and covered with Parafilm. Sterile MEA plugs served as controls. The experiment included 20 replications for each isolate and the control. After 4 weeks, 80% of inoculated plants showed bark necrosis or small cankers, or both, and 20% died. P. funerea was reisolated. No cankers developed in the controls. P. funerea was previously reported in Italy on Thuja orientalis L. (2) and from France on C. leylandii (1), but to our knowledge, this is the first report of the pathogen on C. leylandii in Italy. References: (1) M. Morelet. Rev. Hortic. 227:35, 1982. (2) A. Panconesi and G. Vettori. Inf. Fitopatol. 5:37, 1994.

European Pines May Be Simultaneously Infected by More Than One Species of Heterobasidion.

Heterobasidion annosum (Fr.:Fr.) Bref. sensu lato, one of the most damaging root and butt rot agents on conifers, was recently segregated into three species in Europe based on morphology, intersterility grouping (ISGs), and host preferences (3). These species include: H. annosum (Fr.) Bref. sensu stricto (ISG P) on Pinus, other conifers and some hardwoods; H. parviporum Niemelä & Korhonen (ISG S), primarily on Picea; and H. abietinum Niemelä & Korhonen (ISG F) on Abies. In the summer of 1998, a Swiss stone pine (Pinus cembra L.), growing at 1,900 m in a mixed spruce (Picea) and larch (Larix) forest in the Aosta Valley (northwest Italian Alps), was found infected by H. parviporum and H. annosum sensu stricto. The pine (approximately 14 m tall and at least 75 years old) was without crown symptoms, but the stem, stump, and all the main roots showed internal decay. Disks, 3 to 4 cm thick, were cut consecutively from the roots, stump, and stem, incubated, and examined for conidiophore production. After 8 days, 63 isolates were obtained from all disks taken from the stump and roots, and from disks taken up to 4 m above the collar in the stem. Isolates were identified by polymerase chain reaction (PCR) of mitochondrial and nuclear markers (2) and by sexual compatibility with testers of each European Heterobasidion spp. The stem and one root were colonized by H. parviporum while the other roots and most of the stump was colonized by H. annosum sensu stricto. Somatic incompatibility tests among conspecific isolates suggested that there was only one genet of each species. The coexistence of different Heterobasidion spp. (ISGs) in the same tree has been reported only in Pinus ponderosa Dougl. ex Laws. in California (1) and in Picea abies (L.) Karst. in Europe (4). To our knowledge, this is the first report of H. annosum sensu stricto on P. cembra and of a European pine to be simultaneously infected by more than one species of Heterobasidion. References: (1) M. Garbelotto et al. Phytopathology 86:543, 1996. (2) P. Gonthier et al. Can. J. Bot. 79:1057, 2001. (3) T. Niemelä and K. Korhonen. Taxonomy of the genus Heterobasidion. Pages 27-33 in Heterobasidion annosum, Biology, Ecology, Impact and Control. CAB International, Wallingford, UK, 1998. (4) R. Vasiliauskas and J. Stenlid. Can. J. Forest Res. 28:961, 1998.

First Report of Tubakia dryina in Buds and Shoots of Quercus cerris and Quercus robur.

Tubakia dryina (Sacc.) Sutt. (teleomorph Dicarpella dryina Belisario & Barr) is a widespread leaf pathogen in northern temperate zones and is mainly associated with Quercus spp. During 2000, T. dryina was isolated from seasonal samplings of endophytic fungal communities of Turkey (Q. cerris L.) and English (Q. robur L.) oaks. Samples were taken from healthy and declining trees showing no symptoms of T. dryina in a coppice with saplings in Piedmont (northwestern Italy). Fragments of freshly opened buds (1-year-old shoots) and leaves were surface-sterilized by immersion in 75% ethanol for 1 min, in a NaClO solution (3 to 4% active ingredient) for 3 min, and in 75% ethanol for 30 s, then plated on potato dextrose agar. T. dryina was found in all sampled tissues. Its frequency in buds was higher in healthy trees than in declining trees (≈25 versus 12%; analysis of variance test P < 0.05), whereas no difference was found in shoots. T. dryina was also isolated from asymptomatic leaves, but more often from declining trees than from healthy ones (≈40 versus 10%; P < 0.05). No differences were observed when comparing the two oak species. The fungus was previously reported in buds of Q. nigra L. in North America (2), and it has also been isolated from symptomatic leaves of Q. cerris in a 5-year-old plantation (1). T. dryina was found in other studies in leaves and dead twigs of Q. robur. To our knowledge, this is the first report of T. dryina in buds and shoots of European oak species, suggesting an important role of this fungus as either an endophyte or a latent pathogen associated with oak decline. References: (1) A. Belisario. Plant Dis. 77:647, 1993. (2) Y. C. Zhang and J. T. Walker. Plant Dis. 79:568, 1995.

First Report of Heterobasidion annosum on Native European Pinus cembra.

Heterobasidion annosum (Fr.:Fr.) Bref. is one of the most widespread and damaging root and butt rot agents on conifers. In the summer of 1998, H. annosum was observed for the first time on the Swiss Stone Pine (Pinus cembra L.) in its natural range (1) at 1,900 m in the Aosta Valley in the northwestern Italian Alps. The affected tree was 14 m tall and about 60 years old. It was growing in a mixed spruce (Picea) and larch (Larix) forest severely affected by H. annosum. There were no clear crown symptoms but, after felling, an extensive butt rot was noticed up to 4 m from the collar. The pathogen was isolated from a disk cut at a height of about 40 cm. Its anamorphic form (Spiniger meineckellum (A. Olson) Stalpers) developed on this disk after 8 days of incubation at 20°C. H. annosum was also isolated from the central cylinder of the tree's primary roots and on the other roots down to a diameter of 0.5 cm. Sexual mating tests with single-spore testers for the P, S, and F intersterile groups (ISGs) showed that the isolate belonged to S-ISG. Biomolecular tests on this strain are in progress to confirm this. Reference: (1) T. G. Tutin et al. 1993. Flora Europaea. Cambridge University Press, Cambridge.