A revision of the genus Psathyrella, with a focus on subsection Spadiceogriseae.

Specimens belonging to taxa traditionally assigned to the subsection Spadiceogriseae of the genus Psathyrella were analyzed both morphologically and molecularly. Samples included mainly European collections, selected GenBank accessions, and specimens of various North American taxa described by Smith (1972) and deposited at the Herbarium of the University of Michigan (MICH). Three additional taxa from Africa and Central America were also included. Bayesian and Maximum Likelihood analyses of two loci (ITS and Tef-1α) independently and together supported the monophyletic nature of the subsection Spadiceogriseae, and identified nine statistically supported clades within the subsection. North American and European species often fell within the same clade, suggesting a relatively recent origin of the subsection or human induced intercontinental movement. While this study determines for the first time that the presence of a white veil is diagnostic for the entire subsection, very few morphological traits were associated with individual clades, but clades were often distinctively different in terms of habitat association, suggesting that trophic interactions may have driven the evolution of this group of fungi. Combined, morphological and DNA analyses revealed both expected and unexpected synonymies. The new combinations P. vesiculosa, P. ochrofulva and P. sanjuanensis are proposed, and the new species P. rogersiae is described. New information is provided on the taxonomic status and distribution of several species including P. agrariella, P. albescens, P. atrifolia, P. bivelata, P. fatua, P. kauffmanii, P. aff. kauffmanii, P. incondita, P. infida, P. nitens, P. niveobadia, P. phegophila, P. pseudocorrugis sensu Kits van Waveren, P. subnuda. In total, 13 synonymies were proposed. Based on DNA data, five species of uncertain validity were confirmed as valid, while six species may be ambiguous and may require an in-depth re-analysis. The information gathered in this study was used to generate a key to the species of the subsection Spadiceogriseae.

Detection, Diversity, and Population Dynamics of Waterborne Phytophthora ramorum Populations.

Sudden oak death, the tree disease caused by Phytophthora ramorum, has significant environmental and economic impacts on natural forests on the U.S. west coast, plantations in the United Kingdom, and in the worldwide nursery trade. Stream baiting is vital for monitoring and early detection of the pathogen in high-risk areas and is performed routinely; however, little is known about the nature of water-borne P. ramorum populations. Two drainages in an infested California forest were monitored intensively using stream-baiting for 2 years between 2009 and 2011. Pathogen presence was determined both by isolation and polymerase chain reaction (PCR) from symptomatic bait leaves. Isolates were analyzed using simple sequence repeats to study population dynamics and genetic structure through time. Isolation was successful primarily only during spring conditions, while PCR extended the period of pathogen detection to most of the year. Water populations were extremely diverse, and changed between seasons and years. A few abundant genotypes dominated the water during conditions considered optimal for aerial populations, and matched those dominant in aerial populations. Temporal patterns of genotypic diversification and evenness were identical among aerial, soil, and water populations, indicating that all three substrates are part of the same epidemiological cycle, strongly influenced by rainfall and sporulation on leaves. However, there was structuring between substrates, likely arising due to reduced selection pressure in the water. Additionally, water populations showed wholesale mixing of genotypes without the evident spatial autocorrelation present in leaf and soil populations.

Lineage, Temperature, and Host Species have Interacting Effects on Lesion Development in Phytophthora ramorum.

There are four recognized clonal lineages of the pathogen Phytophthora ramorum. The two major lineages present in North America are NA1 and NA2. With a few exceptions, NA1 is found in natural forest ecosystems and nurseries, and NA2 is generally restricted to nurseries. Isolates from the NA1 and NA2 lineages were used to infect rhododendron, camellia, and California bay laurel in detached leaf assays to study the effects of lineage, temperature, and host on pathogenicity and host susceptibility. Isolates within both lineages were highly variable in their ability to form lesions on each host. There was also a tendency toward reduced lesion size in successive trials, suggesting degeneration of isolates over time. Temperature had a significant effect on lesion size, with a response that varied depending on the host and isolate. Phenotypic differences between lineages appear to be heavily influenced by the representation of isolates used, host, and temperature. The importance of temperature, host, and lineage are discussed with respect to disease management, as well as future range expansions and migrations of the pathogen.

First Report of the NA2 Lineage of Phytophthora ramorum from an Ornamental Rhododendron in the Interior of California.

In July 2012, we collected a rhododendron var. Trilby with twig dieback symptoms in the lower canopy, consistent with the disease "ramorum blight" caused by Phytophthora ramorum. The symptomatic plant had been planted a year earlier to replace a dead rhododendron in a landscape setting in Placer County, California (Lat: 39.036216°; Long: -120.999274°), Sierra Nevada foothills at ~600 m elevation. Isolations yielded a culture with a fast growth rate and overall morphology resembling the P. ramorum NA2 lineage described by Ivors et al. (4). DNA was extracted from the culture as described previously (4) and six SSR loci: MS18, MS39, MS43, MS45, MS64, MS145, were amplified (2,4). Allelic patterns were compared with those of three testers from each of the three lineages NA1, NA2, and EU1 known to be present in ornamental plants in North America, and they unambiguously confirmed the isolate belongs to the NA2 lineage of the pathogen. Although the symptomatic plant was confined to a landscape setting, it had been planted in that location for a year, providing a possible source of inoculum for the surrounding area. This is the first report of P. ramorum from the Sierra Nevada eco-region in the interior of California. It is also the first report of a NA2 isolate from a plant outside of commercial nurseries in California. The mating type of the isolate was not determined, but NA2 isolates are normally A2, the same mating type of NA1 isolates. The only other report of a NA2 isolate found outside of a nursery is from Washington State (1). Although there is no evidence the pathogen may have infected other plants, the infected rhododendron was found at a location situated over 100 km east of the closest known infestation (www.sodmap.org). Additionally, this is the first report of the pathogen outside the coast mountain range of California. Because the three lineages are genetically and phenotypically distinct (3), the escape of NA2 or EU1 isolates, both still absent from plants in natural settings, could have significant implications for California ecosystems. This finding highlights that introductions of P. ramorum via ornamental plants are still possible, in spite of current regulations. References: (1) G. Chastagner et al. Phytopathology 101:S32, 2011. (2) P. P. Croucher et al. Biol. Invasions 15:2281, 2013. (3) N. J. Grünwald et al. Trends Microbiol. 20:131, 2012. (4) K. Ivors et al. Mol. Ecol. 15:1493, 2006.

A First Generation Heterobasidion Hybrid Discovered in Larix lyalli in Montana.

On September 25, 2010, a wood sample was collected from an entirely decayed root ball of an alpine larch (Larix lyallii Parl.), 10 cm in diameter at breast height, recently downed, but still green. No attempts were made to determine whether the decay progressed into the stem. The discovery occurred in a stand in the Bitterroot Mountains, south of Darby, Montana (elev. 2,530 m; 45.893528° N, 114.278322° W). Several adjacent alpine larches were either dead or displayed thin crowns, and an old Heterobasidion basidiocarp was found on the decayed root ball of a neighboring dead tree, suggesting the presence of a root disease pocket. The stand is mature and composed of alpine larch, whitebark pine (Pinus albicaulis Engelm.), and a few subalpine firs (Abies lasiocarpa (Hooker) Nuttall), but only larches were symptomatic. No stumps were visible, and the site is in a designated wilderness area characterized by minimal forest management. Wood chips displaying a white rot with bleached speckles were plated on 2% malt agar, and cultures displaying the typical Heterobasidion anamorph (Spiniger meineckellus) were visible after 7 days. DNA was extracted from two distinct cultures, and the sequences of three nuclear loci, namely the internal transcribed spacer, the elongation factor 1-alpha, and the glyceraldehyde 3-phosphate dehydrogenase, were analyzed. The sequence of the mitochondrial ATPase was also sequenced. All loci were amplified using the primers indicated in Linzer et al. (2). Sequences of all three nuclear loci (GenBank Accession Nos. KF811480 to 82) unequivocally indicated both isolates to be first generation hybrids between H. irregulare (Underw.) Garbel. & Otrosina and H. occidentale Otrosina & Garbel. Cumulatively, sequences were heterozygous at over 40 positions in all three loci, and for the presence of two indels (one in ITS, one in EF 1-alpha). Polymorphisms and indels indicated alleles from both species were present in these heterokaryotic (ploidy n+n) isolates. The mitochondrial ATPase (KF811483 to 84) indicated instead the cytoplasm belonged to H. occidentale, suggesting that species was the first to be established in the infected tree and was either dikaryotized by a basidiopsore of the other species, or subject to nuclear re-assortment through di-mon mating with a genotype of H. irregulare. This is the first report of a Heterobasidion sp. in L. lyalli, and it is the second report of a natural Heterobasidion hybrid in North America (1). This finding indicates Alpine larch may be a host for both Heterobasidion species, as described for pine stumps in California (1). Thus, this conifer may have provided a substrate for the hybridization and interspecific gene introgression documented to have occurred before stumps were generated in high frequency by modern forestry practices (2). References: (1) M. Garbelotto et al. Phytopathology 86:543, 1996. (2) R. Linzer et al. Mol. Phylogenet. Evol. 46:844, 2008.

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.

Population dynamics of aerial and terrestrial populations of Phytophthora ramorum in a California forest under different climatic conditions.

Limited information is available on how soil and leaf populations of the sudden oak death pathogen, Phytophthora ramorum, may differ in their response to changing weather conditions, and their corresponding role in initiating the next disease cycle after unfavorable weather conditions. We sampled and cultured from 425 trees in six sites, three times at the end of a 3-year-long drought and twice during a wet year that followed. Soil was also sampled twice with similar frequency and design used for sampling leaves. Ten microsatellites were used for genetic analyses on cultures from successful isolations. Results demonstrated that incidence of leaf infection tripled at the onset of the first wet period in 3 years in spring 2010, while that of soil populations remained unchanged. Migration of genotypes among sites was low and spatially limited under dry periods but intensity and range of migration of genotypes significantly increased for leaf populations during wet periods. Only leaf genotypes persisted significantly between years, and genotypes present in different substrates distributed differently in soil and leaves. We conclude that epidemics start rapidly at the onset of favorable climatic conditions through highly transmissible leaf genotypes, and that soil populations are transient and may be less epidemiologically relevant than previously thought.

Sequence and simple-sequence repeat analyses of the fungal pathogen Seiridium cardinale indicate California is the most likely source of the cypress canker epidemic for the Mediterranean region.

Seiridium cardinale is the pathogenic fungus of unknown origin responsible for a world pandemic known as cypress canker affecting several species of Cupressaceae in both the Northern and Southern Hemisphere. In this study, a comparative genetic analysis of worldwide populations was performed using sequence analysis of a portion of the ?-tubulin locus and seven polymorphic simple-sequence repeat (SSR) loci on 96 isolates. Sequence analysis identified two distinct ?-tubulin alleles, both present in California. Only one of the two alleles was detected in the Mediterranean basin, while two isolates from the Southern Hemisphere were characterized by the presence of the allele absent from the Mediterranean. SSRs identified a total of 46 multilocus genotypes (MGs): genotypic diversity was always higher in the California population, and calculations of the index of association (I(A)) determined the presence of linkage disequilibrium associated with the absence of sexual reproduction only in the Mediterranean population but not in California. In 50 instances, the same MG was found at great geographic distances, implying a role played by humans in spreading the disease. Network analysis performed on SSR data identified three clusters of MGs: California, Morocco, and the rest of the Mediterranean. Both the Morocco and the Mediterranean clusters were linked to the California cluster. Coalescent analysis identified insignificant migration between California and Italy, as expected in the presence of a single introduction event, and very high migration from Italy into Greece, as expected of an outbreak still in exponential growth phase and starting from an Italian source.

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.

First Report of Seiridium unicorne Causing Bark Cankers on a Monterey Cypress in California.

In June 2009, dieback of distal branches and resin exudation associated with bark lesions were observed in an adult Cupressus macrocarpa tree in Sonoma County, California (Glenn Ellen; 38°21'N, 122°31'W, elevation 233 m). The fungal pathogen, Seiridium unicorne (Cooke and Ellis) Sutton, was obtained by plating fragments of necrotic bark from the margins of branch cankers on potato dextrose agar (PDA). Identification was based on cultural, morphological, and molecular traits (2,3). Colonies on PDA were dense, cottony, off-white at first and then turning pale gray-green, and 2.3 and 4.3 cm in diameter after 1 and 2 weeks of growth at 20°C, respectively. Colonies of the fungus showed a faster radial growth at 20°C than at 25°C. Acervuli were abundantly produced on water agar amended with autoclaved cypress seeds after 2 to 3 weeks at 18°C under a mixture of fluorescent and near UV light. Conidia were six celled (five euseptate), fusiform, 20.9 to 35.2 × 7.11 to 10.57 µm, straight or slightly curved, with four, brown median cells, and with end cells bearing unbranched appendages 2 to 5 µm long. The DNA sequence of a portion of the ß-tubulin locus (GenBank Accession No. HQ678171) revealed a 100% homology with sequences of S. unicorne isolates from Portugal and South Africa, while being clearly distinct from sequences of S. cupressi and S. cardinale isolates (2). Greenhouse stem inoculations were performed by underbark placement of a 3-mm plug taken from the margins of a colony of the fungus grown on PDA. Inoculations were repeated twice in the spring and fall of 2010 on 10 C. macrocarpa saplings grown in pots for 3 years. Three months postinoculation, the pathogen could be successfully reisolated from the edges of 15 to 30 mm long elliptical lesions, present on each one of the inoculated saplings. The observed S. unicorne isolate is atypical because of its shorter appendages compared with the form reported in the literature (2,3). Because of its shorter conidial appendages and in vitro temperature optimum of 18 to 20°C, the fungus described here is similar to an unnamed Coryneum sp. observed by Wagener on C. macrocarpa (4). S. unicorne is a pathogen of many Cupressaceae in Africa, New Zealand, Japan, and some U.S. states (Georgia, South Carolina, Kansas, and Texas) (3), and although it was mentioned in a USDA Plant Quarantine Division report from 1963 as found on cypress in San Francisco (1), it has never been officially reported from California. Since similar disease symptoms were observed on many Cupressaceae in the course of an extensive survey performed in 2009 in California, it may be important to evaluate the relative incidence of S. unicorne compared with that of S. cardinale, a pathogen more commonly reported in association with the disease (4). References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/fungushost/fungushost.cfm , 1/19/2011. (2) P. Krokene et al. Mycologia 96:1352, 2004. (3) N. A. Tisserat. Plant Dis. 75:138, 1991. (4) W. W. Wagener. J. Agric. Res. 58:1, 1939.

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.

Genetic epidemiology of the sudden oak death pathogen Phytophthora ramorum in California.

A total of 669 isolates of Phytophthora ramorum, the pathogen responsible for Sudden Oak Death, were collected from 34 Californian forests and from the ornamental plant-trade. Seven microsatellite markers revealed 82 multilocus genotypes (MGs) of which only three were abundant (>10%). Iteratively collapsing based upon minimum Phi(ST), yielded five meta-samples and five singleton populations. Populations in the same meta-sample were geographically contiguous, with one exception, possibly explained by the trade of infected plants from the same source into different locations. Multidimensional scaling corroborated this clustering and identified nursery populations as genetically most distant from the most recent outbreaks. A minimum-spanning network illustrated the evolutionary relationships among MGs, with common genotypes at the centre and singletons at the extremities; consistent with colonization by a few common genotypes followed by local evolution. Coalescent migration analyses used the original data set and a data set in which local genotypes were collapsed into common ancestral genotypes. Both analyses suggested that meta-samples 1 (Santa Cruz County) and 3 (Sonoma and Marin Counties), act as sources for most of the other forests. The untransformed data set best explains the first phases of the invasion, when the role of novel genotypes may have been minimal, whereas the second analysis best explains migration patterns in later phases of the invasion, when prevalence of novel genotypes was likely to have become more significant. Using this combined approach, we discuss possible migration routes based on our analyses, and compare them to historical and field observations from several case studies.

Evaluation of molecular markers for Phytophthora ramorum detection and identification: testing for specificity using a standardized library of isolates.

Given the importance of Phytophthora ramorum from a regulatory standpoint, it is imperative that molecular markers for pathogen detection are fully tested to evaluate their specificity in detection of the pathogen. In an effort to evaluate 11 reported diagnostic techniques, we assembled a standardized DNA library using accessions from the World Phytophthora Genetic Resource Collection for 315 isolates representing 60 described Phytophthora spp. as well as 11 taxonomically unclassified isolates. These were sent blind to collaborators in seven laboratories to evaluate published diagnostic procedures using conventional (based on internal transcribed spacer [ITS] and cytochrome oxidase gene [cox]1 and 2 spacer regions) and real-time polymerase chain reaction (based on ITS and cox1 and 2 spacer regions as well as beta-tubulin and elicitin genes). Single-strand conformation polymorphism (SSCP) analysis using an automated sequencer for data collection was also evaluated for identification of all species tested. In general, the procedures worked well, with varying levels of specificity observed among the different techniques. With few exceptions, all assays correctly identified all isolates of P. ramorum and low levels of false positives were observed for the mitochondrial cox spacer markers and most of the real-time assays based on nuclear markers (diagnostic specificity between 96.9 and 100%). The highest level of false positives was obtained with the conventional nested ITS procedure; however, this technique is not stand-alone and is used in conjunction with two other assays for diagnostic purposes. The results indicated that using multiple assays improved the accuracy of the results compared with looking at a single assay alone, in particular when the markers represented different genetic loci. The SSCP procedure accurately identified P. ramorum and was helpful in classification of a number of isolates to a species level. With one exception, all procedures accurately identified P. ramorum in blind evaluations of 60 field samples that included examples of plant infection by 11 other Phytophthora spp. The SSCP analysis identified eight of these species, with three identified to a species group.

Microsatellite markers for the diploid basidiomycete fungus Armillaria mellea.

We isolated and characterized 12 microsatellite markers for two North American populations (California, Pennsylvania) of Armillaria mellea, a fungal pathogen responsible for Armillaria root disease of numerous woody plants. Allele frequency ranged from two to nine alleles per locus, and gene diversity ranged from 0.05 to 0.86. Of the 12 loci, eight loci were polymorphic in the California and Pennsylvania populations, and showed no evidence of heterozygote deficiencies or severe linkage disequilibrium. Our results suggest that we have isolated and characterized variable loci to estimate genotypic diversity, gene flow and migration, and to determine population structure of North American A. mellea.

Variation in rates of spore deposition of Fusarium circinatum, the causal agent of pine pitch canker, over a 12-month-period at two locations in Northern California.

Patterns of spore deposition by Fusarium circinatum, the causal agent of pine pitch canker (PPC) of Monterey pine (Pinus radiata) and other conifers, were studied between May 2003 and April 2004 at two sites in Northern California using a novel spore trapping method combined with a real-time polymerase chain reaction (PCR) approach. At each study site, two plots were sampled by placing spore traps at 100 m intervals along transects 600 m in length. The air was sampled continuously by exchanging the spore traps every 2 weeks. The spore deposition rate (DR), ranged from 0 to 1.3 x 10(5) spores m(2). Spores were detected throughout the year, with higher trapping frequencies (TF) during the rainy season (November to April), than during the dry season (May to October). The detection of spores on traps at distances larger than 200 m from any Monterey pine, suggests at least midrange aerial dispersal. Finally, different inoculum loads were associated with trees displaying different levels of disease symptoms, suggesting infectiousness of the pathogen varies as the disease progresses. This study represents one of the first documenting continuous inoculum pressure values over an entire year for a forest pathogen, and provides important epidemiological information that will be invaluable in the development of disease progression models.

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

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

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.

Composting is an effective treatment option for sanitization of Phytophthora ramorum-infected plant material.

AIMS: To determine the effects of heat and composting treatments on the viability of the plant pathogen Phytophthora ramorum grown on both artificial and various natural substrates. METHODS AND RESULTS: Phytophthora ramorum was grown on V8 agar, inoculated on bay laurel leaves (Umbellularia californica) and on woody tissues of coast live oak (Quercus agrifolia). Effects on growth, viability and survival were measured as a result of treatment in ovens and compost piles. Direct plating onto PARP medium and pear-baiting techniques were used to determine post-treatment viability. No P. ramorum was recovered at the end of the composting process, regardless of the isolation technique used. By using a PCR assay designed to detect the DNA of P. ramorum, we were able to conclude the pathogen was absent from mature compost and not merely suppressed or dormant. CONCLUSIONS: Some heat and composting treatments eliminate P. ramorum to lower than detectable levels on all substrates tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Composting is an effective treatment option for sanitization of P. ramorum-infected plant material. Assaying for pathogen viability in compost requires a direct test capable of differentiating between pathogen suppression and pathogen elimination.