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Fungal symbionts occur in all plant tissues, and many aid their host plants with critical functions, including nutrient acquisition, defense against pathogens, and tolerance of abiotic stress. "Core" taxa in the plant mycobiome, defined as fungi present across individuals, populations, or time, may be particularly crucial to plant survival during the challenging seedling stage. However, studies on core seed fungi are limited to individual sampling sites, raising the question of whether core taxa exist across large geographic scales. We addressed this question using both culture-based and culture-free techniques to identify the fungi found in individual seeds collected from nine provenances across the range of coastal Douglas-fir ( Pseudotsuga menziesii var. menziesii ), a foundation tree species in the Pacific Northwest and a globally important timber crop that is propagated commercially by seed. Two key findings emerged: (i) Seed mycobiome composition differed among seed provenances. (ii) Despite variation in the seed mycobiome, we detected four core members, none of which is a known pathogen of Douglas-fir: Trichoderma spp., Hormonema macrosporum, Mucor plumbeus , and Talaromyces rugulosus . Our results support the concept of a core seed microbiome, yet additional work is needed to determine the functional consequences of core taxa for seedling germination, growth, survival, and competition.

Timber durability is often assessed using small wood stakes exposed in direct soil contact, and the assessment generally emphasizes effects on wood rather than organisms involved. Understanding fungal colonization patterns can help identify key decay agents under varying conditions and use these patterns to improve wood protection strategies. Fungal colonization of red alder ( Alnus rubra ), Douglas-fir ( Pseudotsuga menziesii ) heartwood/sapwood, and western redcedar ( Thuja plicata ) field stakes was assessed over 2 y in western Oregon. Spatiotemporal fungal community variations were identified via culturing and DNA sequencing, where 814 isolates were identified from 84 stakes. Forty-six ascomycete genera were identified, with Phialophora, Trichoderma , and Epicoccum species occurring most frequently. Twenty-three basidiomycete genera were identified, with Trametes and Phanerochaete being the most common. Douglas-fir and western redcedar stakes contained the highest and lowest diversity levels, respectively, reflecting natural durability differences of these species. Fungal species abundance was higher below ground than in the above ground and groundline zones, likely reflecting more stable moisture regimes, proximity to soil-based fungi, and potential nutrient migration into wood beneath the soil surface. Ascomycetes were proportionally more abundant early in the exposure period, but basidiomycetes were also observed early in the process, and there appeared to be no consistent colonization pattern.

Swiss needle cast (SNC) is a disease specific to Douglas-fir (Pseudotsuga menziesii) caused by the ascomycete Phaeocryptopus gaeumannii. Here we examine characteristics of the EM fungus community that are potentially useful in predictive models that would monitor forest health. We found that mean EM density (number of colonized root tips/soil core) varied nearly 10-fold among sites of varying levels of SNC, while mean EM fungus species richness (number of species/soil core) varied by about 2.5 times. Strong relationships were found between EM and SNC parameters: EM species richness was positively correlated with both Douglas-fir needle retention (R(2) = 0.93) and EM density (R(2) = 0.65); EM density also was significantly correlated with Douglas-fir needle retention (R(2) = 0.70). These simple characteristics of the EM fungus community could be used to monitor forest health and generate predictive models of site suitability for Douglas-fir. Based on previous findings that normally common EM types were reduced in frequency on sites with severe SNC, we also hypothesized that some EM fungi would be stress tolerant-dominant species. Instead, we found that various fungi were able to form EM with the stressed trees, but none were consistently dominant across samples in the severely diseased areas., (© 2014 by The Mycological Society of America.)

Rhizopogon subgenus Villosuli are the only members of the genus known to form an ectomycorrhizal relationship exclusively with Pseudotsuga. The specificity of this host relationship is unusual in that Rhizopogon is broadly associated with several tree genera within the Pinaceae and relationships with a host genus are typically distributed across Rhizopogon subgenera. Naturally occurring specimens of R. subg. Villosuli have been described only from North American collections, and the unique host relationship with Pseudotsuga is demonstrated only for Rhizopogon associated with P. menziesii (Douglas-fir), the dominant species of Pseudotsuga in North America. Species of Pseudotsuga are naturally distributed around the northern Pacific Rim, and Rhizopogon associates of other Pseudotsuga spp. are not yet described. Here we present the results of field sampling conducted in P. japonica forests throughout the Japanese archipelago and describe Rhizopogon togasawariana sp. nov., which occurs in ectomycorrhizal association with P. japonica. Placement of this new species within R. subg. Villosuli is supported by morphological and molecular phylogenetic analysis, and its implications to Pseudotsuga-Rhizopogon biogeography are discussed.

Using spatial autocorrelation analysis, we examined the within-population genetic structure of Rhizopogon vinicolor and R. vesiculosus, two hypogeous ectomycorrhizal (EM) species that are sympatric sister taxa known to differ in their clonal structure. We collected 121 sporocarps and 482 tuberculate EM of both species from a 20 ha forest stand dominated by Douglas-fir (Pseudotsuga menziesii). Field collections were identified to species with restriction fragment length polymorphism analysis of the nuclear ribosomal internal transcribed spacer. Five and six microsatellite markers were used to characterize the genetic diversity of EM and sporocarp samples from R. vesiculosus and R. vinicolor respectively. After correcting for genet structure, spatial autocorrelation analyses of the EM samples were used to test the null hypothesis that multilocus genotypes characterized from each species were randomly distributed within the study area. We detected positive and statistically significant fine-scale genetic structure up to 120 m within the R. vesiculosus sample. In contrast, no spatial genetic structure was evident for R. vinicolor, indicating that the genotypes characterized for this species were randomly distributed throughout the study area. Differences in statistical power or the nuclear count of basidiospores are unlikely agents of the genetic patterns observed. Our results suggest that differences in reproductive output or competitive ability may act individually or in combination to create clusters of similar genotypes for R. vesiculosus throughout the study area.

Kalapuya is described as a new, monotypic truffle genus in the Morchellaceae known only from the Pacific northwestern United States. Its relationship to other hypogeous genera within Morchellaceae is explored by phylogenetic analysis of the ribosomal LSU and EF1alpha protein coding region. The type species, K. brunnea, occurs in Douglas-fir forests up to about 50 y old on the west slope of the Cascade Range in Oregon and in the Coastal Ranges of Oregon and northern California. It has a roughened, warty, reddish brown to brown peridium, a solid whitish gleba that develops grayish brown mottling as the spores mature, and produces a cheesy-garlicky odor at maturity. Its smooth, ellipsoid spores resemble those of Morchella spp. but are much larger. The four hypogeous genera known in the Morchellaceae, Kalapuya, Fischerula, Imaia and Leucangium, are distinct from the epigeous genera Morchella and Verpa, but it is uncertain whether they resulted from a single transition to a hypogeous fruiting habit or from multiple independent transitions. Kalapuya, locally known as the Oregon brown truffle, has been commercially harvested for culinary use.

Germinating ascospores of Phaeocryptopus gaeumannii produce suprastomatal appressoria from which penetration pegs enter needles. Initial infection occurs between late May and early Jul and coincides with budbreak and shoot elongation. Colonization within needles is exclusively intercellular and increases continuously during Jul-May. No intracellular hyphae or haustoria were observed, but hyphae closely appressed to mesophyll and palisade cell walls are abundant by 3-5 mo after initial infection. Pseudothecial primordia begin to form in epistomatal chambers Oct-Apr, 4-9 mo after initial infection. Pseudothecial primordia developing in the epistomatal chamber are connected to the endophytic thallus by specialized cells in the substomatal chamber that have thickened apical walls and resemble phialides but are not involved in asexual reproduction. The apical wall thickenings instead appear to function as reinforcement against the turgor pressure of the guard cells, allowing cytoplasmic continuity to be maintained between the developing pseudothecium and vegetative hyphae within the needle. Concurrent with the formation of pseudothecial primorida, epiphytic hyphae emerge from the periphery of developing pseudothecia, grow across the needle surface, form numerous anastomoses and reenter the needle by producing appressoria above unoccupied stomata. Epiphytic hyphae and their associated appressoria gradually become more abundant during Oct-Jan.

Phaeocryptopus gaeumannii, causal agent of the Douglas-fir foliar disease Swiss needle cast, is the only known pathogenic species of the genus. Current classifications place Phaeocryptopus in the Venturiaceae (Pleosporales), typified by the apple-scab pathogen Venturia inaequalis. All core members of this family have hyphomycetous anamorphs. We sought to confirm these relationships by means of phylogenetic analyses of the small (SSU) and large (LSU) subunits and internal transcribed spacer (ITS) region of nuclear ribosomal gene sequences (nrDNA). Analyses indicated that both the genus Phaeocryptopus and the family Venturiaceae, as currently defined, are unnatural groups. Phaeocryptopus nudus, type of the genus, is aligned in the Dothioraceae (Dothideales) and P. gaeumannii in the Mycosphaerellaceae (Capnodiales) near species of Mycosphaerella and Rasutoria. Core representatives of Venturiaceae formed an unambiguous clade but ordinal placement was unresolved. The family apparently is not included in the Pleosporales, Dothideales, Myriangiales or Capnodiales. Coelomycetous Rhizosphaera form-species are accepted generally as anamorphic states of Phaeocryptopus, however the relationship never has been established conclusively. Species of Rhizosphaera are closely related to P. nudus but not to P. gaeumannii, supporting an anamorph-teleomorph connection between Rhizosphaera and Phaeocryptopus and providing further evidence that P. gaeumanii is not congeneric with P. nudus.

A survey of the genetic diversity and population structure of the Douglas-fir Swiss needle cast pathogen Phaeocryptopus gaeumannii was conducted with single-strand conformational polymorphisms (SSCP) to screen for variability in mitochondrial and nuclear housekeeping genes. Thirty host populations representing the natural range of Douglas-fir as well as locations where the tree was planted as an exotic were sampled. Sequencing of SSCP variants revealed that the method accurately detected both single nucleotide and indel polymorphisms. Sequence information was used to construct multilocus gene genealogies and to test various hypotheses of recombination (outcrossing) and clonality (selfing). We found that P. gaeumannii in the region of Oregon's Swiss needle cast epidemic exhibits strong multilocus gametic phase disequilibrium and is subdivided into two reproductively isolated sympatric lineages. Low genotypic diversity together with the presence of overrepresented genotypes in both lineages suggests a predominantly selfing reproductive mode. Genotypes of one lineage were found in isolates from a widespread geographic distribution, occurring throughout much of the Pacific Northwest as well as nonindigenous populations abroad that have historical reports of disease. Genotypes of the second lineage were detected only in isolates from Oregon's coastal region. Within the main epidemic area, abundance of this second lineage in young plantations appeared to be correlated with disease severity.

We examined the within-population genetic structure of the Pacific golden chanterelle (Cantharellus formosus) in a 50 y old forest stand dominated by Douglas-fir (Pseudotsuga menziesii) and western hemlock (Tsuga heterophylla) with spatial autocorrelation analysis. We tested the null hypothesis that multilocus genotypes possessed by chanterelle genets were randomly distributed within the study area. Fruit bodies from 203 C. formosus genets were collected from a 50 ha study plot. One hundred six unique multilocus genotypes were identified after scoring these collections at five microsatellite loci. Statistically significant positive spatial autocorrelation was detected indicating the presence of fine-scale genetic structure within the area. Repeated autocorrelation analyses with varied minimum distance classes (50-500 m) detected positive spatial genetic structure up to 400 m. Therefore nonrandom evolutionary processes (e.g., isolation by distance) can cause fine-scale genetic structure in C. formosus. The implications of this research for future broad-scale population studies of this species are that population samples should be separated by at least 400 m to be considered statistically independent. Sampling designs that account for fine-scale genetic structure will better characterize heterogeneity distributed across the landscape by avoiding the effects of pseudo replication.