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Ectomycorrhizal (ECM) fungi distribute tree-derived carbon (C) via belowground hyphal networks in forest ecosystems. Here, we asked the following: (1) Is C transferred belowground to a neighboring tree retained in fungal structures or transported within the recipient tree? (2) Is the overlap of ectomycorrhizal fungi in mycorrhizal networks related to the amount of belowground C transfer? We used potted sapling pairs of European beech (Fagus sylvatica) and North-American Douglas-fir (Pseudotsuga menziesii) for 13 CO 2 pulse-labeling. We compared 13 C transfer from beech (donor) to either beech or Douglas-fir (recipient) and identified the ECM species. We measured the 13 C enrichment in soil, plant tissues, and ECM fractions of fungal-containing parts and plant transport tissues. In recipients, only fungal-containing tissue of ectomycorrhizas was significantly enriched in 13 C and not the plant tissue. Douglas-fir recipients shared on average one ECM species with donors and had a lower 13 C enrichment than beech recipients, which shared on average three species with donors. Our results support that recently assimilated C transferred belowground is shared among fungi colonizing tree roots but not among trees. In mixed forests with beech and Douglas-fir, the links for C movement might be hampered due to low mycorrhizal overlap with consequences for soil C cycling., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Understanding the genetic basis of how plants defend against pathogens is important to monitor and maintain resilient tree populations. Swiss needle cast (SNC) and Rhabdocline needle cast (RNC) epidemics are responsible for major damage of forest ecosystems in North America. Here we investigate the genetic architecture of tolerance and resistance to needle cast diseases in Douglas-fir (Pseudotsuga menziesii) caused by two fungal pathogens: SNC caused by Nothophaeocryptopus gaeumannii, and RNC caused by Rhabdocline pseudotsugae. We performed case-control genome-wide association analyses and found disease resistance and tolerance in Douglas-fir to be polygenic and under strong selection. We show that stomatal regulation as well as ethylene and jasmonic acid pathways are important for resisting SNC infection, and secondary metabolite pathways play a role in tolerating SNC once the plant is infected. We identify a major transcriptional regulator of plant defense, ERF1, as the top candidate for RNC resistance. Our findings shed light on the highly polygenic architectures underlying fungal disease resistance and tolerance and have important implications for forestry and conservation as the climate changes., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

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.

Six new 6-isopentylsphaeropsidones, strobiloscyphones A-F ( 1 - 6 ), and a new hexadecanoic acid, (2 Z ,4 E ,6 E )-8,9-dihydroxy-10-oxohexadeca-2,4,6-trienoic acid ( 7 ), together with sphaeropsidone ( 8 ) and its known synthetic analogue 5-dehydrosphaeropsidone ( 9 ) were isolated from Strobiloscypha sp. AZ0266, a fungus inhabiting the leaf litter of Douglas fir ( Pseudotsuga menziesii ). The structures of 1 - 7 were established on the basis of their high-resolution mass and 1D and 2D NMR spectroscopic data, and their relative and/or absolute configurations were determined by NOE, comparison of experimental and calculated ECD spectra, and application of the modified Mosher's ester method. Of these, strobiloscyphone F ( 6 ) contains a novel highly oxygenated tetracyclic oxireno-octahydrodibenzofuran ring system. Natural products 1 , 6 , and 9 and the semisynthetic analogue 12 derived from 8 exhibited cytotoxic activity, whereas 9 and 12 showed antimicrobial activity. Possible biosynthetic pathways to 1 - 6 , 8 , and 9 are proposed.

The emergence of Phytophthora pluvialis as a foliar pathogen of Douglas fir in New Zealand and the Pacific Northwest United States has raised questions about its interaction with the widespread Swiss needle cast (SNC) disease. During Spring 2017, we repeatedly sampled 30 trees along an environmental gradient in each region and 292 additional trees in a longitudinal transect to assess the P. pluvialis epidemic and the association between P. pluvialis and Nothophaeocryptopus gaeumannii , which are causal agents of SNC. Both pathogens were consistently more abundant in the host's exotic environment in New Zealand. In both areas, the two pathogens co-exist in different spatial scales for regions and needles. The relative abundance of both pathogens was negatively correlated in the Pacific Northwest, where both presumably have co-existed for longer. Our findings confirmed the interaction of P. pluvialis and N. gaeumannii as foliar pathogens of Douglas fir and suggest a within-site spatial variation in the Pacific Northwest.

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 is a foliar disease of Douglas-fir (Pseudotsuga menziesii) that results in premature foliage loss and reduced growth. The causal fungus, Nothophaeocryptopus gaeumannii, was first detected in New Zealand in 1959 and spread throughout the North and South Islands over the following decades. The contemporary genetic structure of the N. gaeumannii population in New Zealand was assessed by analyzing 468 multilocus SSR genotypes (MLGs) from 2,085 N. gaeumannii isolates collected from 32 sites in the North and South Islands. Overall diversity was lower than that reported from native N. gaeumannii populations in the northwestern United States, which was expected given that N. gaeumannii is introduced in New Zealand. Linkage disequilibrium was significantly higher than expected under random mating, suggesting that population structure is clonal. Populations of N. gaeumannii in the North and South Islands were weakly differentiated, and the isolates collected from sites within the islands were moderately differentiated. This suggests that gene flow has occurred between the N. gaeumannii populations in the North and South Islands, and between the local N. gaeumannii populations within each island. Eighteen isolates of N. gaeumannii Lineage 2, which has previously been reported only from western Oregon, were recovered from two sites in the North Island and four sites in the South Island. The most likely explanation for the contemporary distribution of N. gaeumannii in New Zealand is that it was introduced on infected live seedlings through the forestry or ornamental nursery trade, as the fungus is neither seed borne nor saprobic, and the observed population structure is not consistent with a stochastic intercontinental dispersal event.

Suillus lakei is an ectomycorrhizal fungus native to North America and known in Europe, South America, and New Zealand. This contribution aims to illustrate the worldwide biogeography of S. lakei based on sporocarp records. Species distribution modeling was used to assess the suitable niche distribution of S. lakei, based on the climatic variables as well as distribution of its ectomycorrhizal partner, Douglas fir. In general, distribution of suitable niches of S. lakei greatly overlaps with the distribution of Douglas fir in North America. By spatial distribution modeling, we found that the precipitation of the coldest quarters, isothermality, and annual mean temperature are important factors influencing the potential distribution of S. lakei. Nevertheless, the most crucial factor limiting expansion of S. lakei in its invasion range is Douglas fir occurrence. This factor reached an 86.4% contribution for the S. lakei species distribution model. Additionally, we compare the aboveground and belowground presence of S. lakei based on surveys in the field. Our study shows that even extremely low abundance of ectomycorrhizas can open the possibility of using an ectomycorrhiza survey for their quantification as a good indicator of the presence of S. lakei in field conditions. Both sporocarps and ectomycorrhizas occurred only in gardens, where Douglas fir seedlings were outplanted at the beginning of the 1990s as an ornamental plant. Presumably, international trade of ornamental plants was one possible route of introduction of S. lakei to Poland.

Chinese Douglas-fir (Pseudotsuga sinensis) is an endangered Pinaceae species found in several isolated regions of China. Although soil spore banks of ectomycorrhizal (ECM) fungi can play an important role in seedling establishment after disturbance, such as in the well-known North American relative (Pseudotsuga menziesii), we have no information about soil spore bank communities in relict forests of Chinese Douglas-fir. We conducted bioassays of 73 soil samples collected from three Chinese Douglas-fir forests, using North American Douglas-fir as bait seedlings, and identified 19 species of ECM fungi. The observed spore bank communities were significantly different from those found in ECM fungi on the roots of resident trees at the same sites (p = 0.02). The levels of potassium (K), nitrogen (N), organic matter, and the pH of soil were the dominant factors shaping spore bank community structure. A new Rhizopogon species was the most dominant species in the spore banks. Specifically, at a site on Sanqing Mountain, 22 of the 57 surviving bioassay seedlings (representing 21 of the 23 soil samples) were colonized by this species. ECM fungal richness significantly affected the growth of bioassay seedlings (R 2  = 0.20, p = 0.007). Growth was significantly improved in seedlings colonized by Rhizopogon or Meliniomyces species compared with uncolonized seedlings. Considering its specificity to Chinese Douglas-fir, predominance in the soil spore banks, and positive effect on host growth, this new Rhizopogon species could play critical roles in seedling establishment and forest regeneration of endangered Chinese Douglas-fir.

Processes governing the fixation, partitioning, and mineralization of carbon in soils are under increasing scrutiny as we develop a more comprehensive understanding of global carbon cycling. Here we examined fixation by Douglas-fir seedlings and transfer to associated ectomycorrhizal fungi, soil microbes, and full-sibling or nonsibling neighbouring seedlings. Stable isotope probing with 99% 13 C-CO 2 was applied to trace 13 C-labelled photosynthate throughout plants, fungi, and soil microbes in an experiment designed to assess the effect of relatedness on 13 C transfer between plant pairs. The fixation and transfer of the 13 C label to plant, fungal, and soil microbial tissue was examined in biomass and phospholipid fatty acids. After a 6 d chase period, c. 26.8% of the 13 C remaining in the system was translocated below ground. Enrichment was proportionally greatest in ectomycorrhizal biomass. The presence of mesh barriers (0.5 or 35 μm) between seedlings did not restrict 13 C transfer. Fungi were the primary recipients of 13 C-labelled photosynthate throughout the system, representing 60-70% of total 13 C-enriched phospholipids. Full-sibling pairs exhibited significantly greater 13 C transfer to recipient roots in two of four Douglas-fir families, representing three- and fourfold increases (+ c. 4 μg excess 13 C) compared with nonsibling pairs. The existence of a root/mycorrhizal exudation-hyphal uptake pathway was supported., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Many truffle species in the genus Tuber are endemic to North America. Some of these have commercial value such as Tuber oregonense and Tuber gibbosum, commonly known as Oregon white truffles. Most of what is known about the ecology of these truffles comes from observational data. These truffle species form ectomycorrhizas with Douglas-fir (Pseudotsuga menziesii) and sometimes fruit abundantly in early successional forest regrowth. The goal of this study was to characterize fungal communities and soils associated with truffle-producing Douglas-fir sites. We extracted DNA from roots of five trees at four different truffle-producing Douglas-fir sites (n = 20). We amplified the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (nrDNA) and sequenced amplicons with 454 pyrosequencing. After quality filtering, we assembled 15,713 sequences into 150 fungal operational taxonomic units (OTUs). Pezizomycetes (Tuber and Pyronemataceae) were the most abundant taxa detected followed by Helotiales. Agaricomycetes represented most by Thelephoraceae, Russulaceae, and Inocybaceae were also abundant. A total of five Tuber species were detected. T. oregonense was the most abundant OTU, followed by T. gibbosum and Wilcoxina mikolae. Fungal root endophytes were also detected and well represented by Chalara and Phialocephala spp. Fungal community structure and soil chemistry differed between sites. This study represents the first characterization of the fungal communities in Douglas-fir stands producing Oregon white truffles. We found that Tuber species can be dominant ectomycorrhizal symbionts of Douglas-fir. Truffle fungi are also important in forest health, food webs, and as a non-timber forest resource that can contribute to rural economies.

Soil depth partitioning is thought to promote the diversity of ectomycorrhizal (EM) fungal communities, but little is known about whether it is controlled by abiotic or biotic factors. In three bioassay experiments, we tested the role of vertical soil heterogeneity in determining the distributions and competitive outcomes of the EM sister species Rhizopogon vinicolor and Rhizopogon vesiculosus. We planted Pseudotsuga menziesii seedlings into soils that were either a homogenized mix of upper and lower depths or vertically stratified combinations mimicking natural field conditions. We found that both species colonized the upper or lower soil depths in the absence of competition, suggesting that their distributions were not limited by abiotic edaphic factors. In competition within homogeneous soils, R. vesiculosus completely excluded colonization by R. vinicolor, but R. vinicolor was able to persist when soils were stratified. The amount of colonization by R. vinicolor in the stratified soils was also significantly correlated with the number of multilocus genotypes present. Taken together, our findings suggest that the differential vertical distributions of R. vinicolor and R. vesiculosus in natural settings are probably attributable to competition rather than edaphic specialization, but that soil heterogeneity may play a key role in promoting EM fungal diversity., (© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)

Rhizopogon vesiculosus and Rhizopogon vinicolor are sister species of ectomycorrhizal fungi that associate exclusively with Douglas-fir (DF). They form tuberculate mycorrhizas and they can be easily distinguished using molecular tools. We are not aware of studies relating their relative abundance in forests with different age classes. Our objective was to determine whether a change in the number or relative abundance of R. vesiculosus and R. vinicolor tubercules and genotypes was related to a change in the percent of DF in a regenerating phase (<50 years old). R. vesiculosus and R. vinicolor were located by excavating tuberculate mycorrhizas from the forest floor. A DNA Alu1 digest was used to distinguish between the two species. Microsatellite markers were used to identify genotypes. The number of R. vesiculosus tubercules correlated positively with an increasing proportion of DF in a regenerating phase, while the number of R. vinicolor tubercules was similar across all forest age structures. The number of R. vesiculosus genotypes did not correlate with forest age structure, whereas the number of R. vinicolor genotypes showed a negative relationship with an increasing proportion of DF in a regenerating phase. When the numbers of R. vesiculosus tubercules and genotypes were expressed as a relative abundance of the two species, there was a positive correlation with an increasing proportion of DF in a regenerating phase for both genotypes and tubercules. Our results suggest that the degree of DF regeneration or ecosystem factors related to DF regeneration affect the population dynamics of R. vesiculosus and R. vinicolor differently.

Ectomycorrhizal (EcM) fungal communities may be shaped by both deterministic and stochastic processes, potentially influencing ecosystem development and function. We evaluated community assembly processes for EcM fungi of Pseudotsuga menziesii among 12 sites up to 400 km apart in southwest British Columbia (Canada) by investigating species turnover (β-diversity) in relation to soil nitrogen (N) availability and physical distance. We then examined functional traits for an N-related niche by quantifying net fluxes of NH4+, NO3- and protons on excised root tips from three contrasting sites using a microelectrode ion flux measurement system. EcM fungal communities were well aligned with soil N availability and pH, with no effect of site proximity (distance-decay curve) on species assemblages. Species turnover was significant (β(1/2) = 1.48) along soil N gradients, with many more Tomentella species on high N than low N soils, in contrast to Cortinarius species. Ammonium uptake was greatest in the spring on the medium and rich sites and averaged over 190 nmol/m(2)/s for Tomentella species. The lowest uptake rates of NH4+ were by nonmycorrhizal roots of axenically grown seedlings (10 nmol/m(2)/s), followed by Cortinarius species (60 nmol/m(2)/s). EcM roots from all sites displayed only marginal uptake of nitrate (8.3 nmol/m(2)/s). These results suggest NH4+ uptake capacity is an important functional trait influencing the assembly of EcM fungal communities. The diversity of EcM fungal species across the region arguably provides critical belowground adaptations to organic and inorganic N supply that are integral to temperate rainforest ecology., (© 2015 John Wiley & Sons Ltd.)

Climatic and land use changes have significant consequences for the distribution of tree species, both through natural dispersal processes and following management prescriptions. Responses to these changes will be expressed most strongly in seedlings near current species range boundaries. In northern temperate forest ecosystems, where changes are already being observed, ectomycorrhizal fungi contribute significantly to successful tree establishment. We hypothesised that communities of fungal symbionts might therefore play a role in facilitating, or limiting, host seedling range expansion. To test this hypothesis, ectomycorrhizal communities of interior Douglas-fir and interior lodgepole pine seedlings were analysed in a common greenhouse environment following growth in five soils collected along an ecosystem gradient. Currently, Douglas-fir's natural distribution encompasses three of the five soils, whereas lodgepole pine's extends much further north. Host filtering was evident amongst the 29 fungal species encountered: 7 were shared, 9 exclusive to Douglas-fir and 13 exclusive to lodgepole pine. Seedlings of both host species formed symbioses with each soil fungal community, thus Douglas-fir did so even where those soils came from outside its current distribution. However, these latter communities displayed significant taxonomic and functional differences to those found within the host distribution, indicative of habitat filtering. In contrast, lodgepole pine fungal communities displayed high functional similarity across the soil gradient. Taxonomic and/or functional shifts in Douglas-fir fungal communities may prove ecologically significant during the predicted northward migration of this species; especially in combination with changes in climate and management operations, such as seed transfer across geographical regions for forestry purposes.

Biological invasions are a rapidly increasing driver of global change, yet fundamental gaps remain in our understanding of the factors determining the success or extent of invasions. For example, although most woody plant species depend on belowground mutualists such as mycorrhizal fungi and nitrogen-fixing bacteria, the relative importance of these mutualisms in conferring invasion success is unresolved. Here, we describe how neighborhood context (identity of nearby tree species) affects the formation of belowground ectomycorrhizal partnerships between fungi and seedlings of a widespread invasive tree species, Pseudotsuga menziesii (Douglas-fir), in New Zealand. We found that the formation of mycorrhizal partnerships, the composition of the fungal species involved in these partnerships, and the origin of the fungi (co-invading or native to New Zealand) all depend on neighborhood context. Our data suggest that nearby ectomycorrhizal host trees act as both a reservoir of fungal inoculum and a carbon source for late-successional and native fungi. By facilitating mycorrhization of P. menziesii seedlings, adult trees may alleviate mycorrhizal limitation at the P. menziesii invasion front. These results highlight the importance of studying biological invasions across multiple ecological settings to understand establishment success and invasion speed.

Separating edaphic impacts on tree distributions from those of climate and geography is notoriously difficult. Aboveground and belowground factors play important roles, and determining their relative contribution to tree success will greatly assist in refining predictive models and forestry strategies in a changing climate. In a common glasshouse, seedlings of interior Douglas-fir (Pseudotsuga menziesii var. glauca) from multiple populations were grown in multiple forest soils. Fungicide was applied to half of the seedlings to separate soil fungal and nonfungal impacts on seedling performance. Soils of varying geographic and climatic distance from seed origin were compared, using a transfer function approach. Seedling height and biomass were optimized following seed transfer into drier soils, whereas survival was optimized when elevation transfer was minimised. Fungicide application reduced ectomycorrhizal root colonization by c. 50%, with treated seedlings exhibiting greater survival but reduced biomass. Local adaptation of Douglas-fir populations to soils was mediated by soil fungi to some extent in 56% of soil origin by response variable combinations. Mediation by edaphic factors in general occurred in 81% of combinations. Soil biota, hitherto unaccounted for in climate models, interacts with biogeography to influence plant ranges in a changing climate., (© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)

Climatic adaptations are the foundation of conifer genecology, but populations also display variation in traits for nitrogen (N) utilization, along with some heritable specificity for ectomycorrhizal fungi (EMF). We examined soil and EMF influences on assisted migration of Douglas-fir (Pseudotsuga menziesii var. menziesii) by comparing two contrasting maritime populations planted up to 400 km northward in southwestern British Columbia. Soil N availability and host N status (via δ(15) N) were assessed across 12 maritime test sites, whereas EMF on local and introduced hosts were quantified by morphotyping with molecular analysis. Climatic transfer effects were only significant with soil N concentrations of test sites as a covariate, and illustrated how height growth was compromised for populations originating from relatively dry or cool maritime environments. We also found evidence for EMF maladaptation, where height declined by up to 15% with the extent of dissimilarity in EMF communities of southern populations relative to local hosts. The results demonstrate how geographic structure in belowground environments can contribute to conifer genecology. Differences in the inherent growth potential of conifers may be partly related to nutritional adaptations arising under native soil fertility, and optimization of this growth potential likely requires close affiliation with local EMF communities., (© 2015 Province of British Columbia. New Phytologist © 2015 New Phytologist Trust.)

The coordinated transcriptomic responses of both mutualistic ectomycorrhizal (ECM) fungi and their hosts during the establishment of symbiosis are not well-understood. This study characterizes the transcriptomic alterations of the ECM fungus Laccaria bicolor during different colonization stages on two hosts (Populus trichocarpa and Pseudotsuga menziesii) and compares this to the transcriptomic variations of P. trichocarpa across the same time-points. A large number of L. bicolor genes (≥ 8,000) were significantly regulated at the transcriptional level in at least one stage of colonization. From our data, we identify 1,249 genes that we hypothesize is the 'core' gene regulon necessary for the mutualistic interaction between L. bicolor and its host plants. We further identify a group of 1,210 genes that are regulated in a host-specific manner. This variable regulon encodes a number of genes coding for proteases and xenobiotic efflux transporters that we hypothesize act to counter chemical-based defenses simultaneously activated at the transcriptomic level in P. trichocarpa. The transcriptional response of the host plant P. trichocarpa consisted of differential waves of gene regulation related to signaling perception and transduction, defense response, and the induction of nutrient transfer in P. trichocarpa tissues. This study, therefore, gives fresh insight into the shifting transcriptomic landscape in both the colonizing fungus and its host and the different strategies employed by both partners in orchestrating a mutualistic interaction.

Background: Phellinus sulphurascens is a fungal pathogen that causes laminar root rot in conifers, one of the most damaging root diseases in western North America. Despite its importance as a forest pathogen, this fungus is still poorly studied at the genomic level. An understanding of the molecular events involved in establishment of the disease should help to develop new methods for control of this disease., Results: We generated over 4600 expressed sequence tags from two cDNA libraries constructed using either mycelia grown on cellophane sheets and exposed to Douglas-fir roots or tissues from P. sulphurascens-infected Douglas-fir roots. A total of 890 unique genes were identified from the two libraries, and functional classification of 636 of these genes was possible using the Functional Catalogue (FunCat) annotation scheme. cDNAs were identified that encoded 79 potential virulence factors, including numerous genes implicated in virulence in a variety of phytopathogenic fungi. Many of these putative virulence factors were also among 82 genes identified as encoding putatively secreted proteins. The expression patterns of 86 selected fungal genes over 7 days of infection of Douglas-fir were examined using real-time PCR, and those significantly up-regulated included rhamnogalacturonan acetylesterase, 1,4-benzoquinone reductase, a cyclophilin, a glucoamylase, 3 hydrophobins, a lipase, a serine carboxypeptidase, a putative Ran-binding protein, and two unknown putatively secreted proteins called 1 J04 and 2 J12. Significantly down-regulated genes included a manganese-superoxide dismutase, two metalloproteases, and an unknown putatively secreted protein called Ps0058., Conclusions: This first collection of Phellinus sulphurascens EST sequences and its annotation provide an important resource for future research aimed at understanding key virulence factors of this forest pathogen. We examined the expression patterns of numerous fungal genes with potential roles in virulence, and found a collection of functionally diverse genes that are significantly up- or down-regulated during infection of Douglas-fir seedling roots by P. sulphurascens.

Swiss needle cast (SNC) is a fungal disease of Douglas-fir (Pseudotsuga menziesii) that has recently become prevalent in coastal areas of the Pacific Northwest. We used growth measurements and stable isotopes of carbon and oxygen in tree-rings of Douglas-fir and a non-susceptible reference species (western hemlock, Tsuga heterophylla) to evaluate their use as proxies for variation in past SNC infection, particularly in relation to potential explanatory climate factors. We sampled trees from an Oregon site where a fungicide trial took place from 1996 to 2000, which enabled the comparison of stable isotope values between trees with and without disease. Carbon stable isotope discrimination (Δ(13)C) of treated Douglas-fir tree-rings was greater than that of untreated Douglas-fir tree-rings during the fungicide treatment period. Both annual growth and tree-ring Δ(13)C increased with treatment such that treated Douglas-fir had values similar to co-occurring western hemlock during the treatment period. There was no difference in the tree-ring oxygen stable isotope ratio between treated and untreated Douglas-fir. Tree-ring Δ(13)C of diseased Douglas-fir was negatively correlated with relative humidity during the two previous summers, consistent with increased leaf colonization by SNC under high humidity conditions that leads to greater disease severity in following years., (© 2013 John Wiley & Sons Ltd.)

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.)

Alnus trees associate with ectomycorrhizal (ECM) fungi and nitrogen-fixing Frankia bacteria and, although their ECM fungal communities are uncommonly host specific and species poor, it is unclear whether the functioning of Alnus ECM fungal symbionts differs from that of other ECM hosts. We used exoenzyme root tip assays and molecular identification to test whether ECM fungi on Alnus rubra differed in their ability to access organic phosphorus (P) and nitrogen (N) when compared with ECM fungi on the non-Frankia host Pseudotsuga menziesii. At the community level, potential acid phosphatase (AP) activity of ECM fungal root tips from A. rubra was significantly higher than that from P. menziesii, whereas potential leucine aminopeptidase (LA) activity was significantly lower for A. rubra root tips at one of the two sites. At the individual species level, there was no clear relationship between ECM fungal relative root tip abundance and relative AP or LA enzyme activities on either host. Our results are consistent with the hypothesis that ECM fungal communities associated with Alnus trees have enhanced organic P acquisition abilities relative to non-Frankia ECM hosts. This shift, in combination with the chemical conditions present in Alnus forest soils, may drive the atypical structure of Alnus ECM fungal communities., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

Stored non-structural carbohydrates (NSCs) could play an important role in tree survival in the face of a changing climate and associated stress-related mortality. We explored the effects of the stomata-blocking and defoliating fungal disease called Swiss needle cast on Douglas-fir carbohydrate reserves and growth to evaluate the extent to which NSCs can be mobilized under natural conditions of low water stress and restricted carbon supply in relation to potential demands for growth. We analyzed the concentrations of starch, sucrose, glucose and fructose in foliage, twig wood and trunk sapwood of 15 co-occurring Douglas-fir trees expressing a gradient of Swiss needle cast symptom severity quantified as previous-year functional foliage mass. Growth (mean basal area increment, BAI) decreased by ∼80% and trunk NSC concentration decreased by 60% with decreasing functional foliage mass. The ratio of relative changes in NSC concentration and BAI, an index of the relative priority of storage versus growth, more than doubled with increasing disease severity. In contrast, twig and foliage NSC concentrations remained nearly constant with decreasing functional foliage mass. These results suggest that under disease-induced reductions in carbon supply, Douglas-fir trees retain NSCs (either actively or due to sequestration) at the expense of trunk radial growth. The crown retains the highest concentrations of NSC, presumably to maintain foliage growth and shoot extension in the spring, partially compensating for rapid foliage loss in the summer and fall.

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.

Host identity is among the most important factors in structuring ectomycorrhizal (ECM) fungal communities. Both host-fungal coevolution and host shifts can account for the observed host effect, but their relative significance in ECM fungal communities is not well understood. To investigate these two host-related mechanisms, we used relict forests of Pseudotsuga japonica, which is an endangered endemic species in Japan. As with other Asian Pseudotsuga species, P. japonica has been isolated from North American Pseudotsuga spp. since the Oligocene and has evolved independently as a warm-temperate species. We collected 100 soil samples from four major localities in which P. japonica was mixed with other conifers and broadleaf trees. ECM tips in the soil samples were subjected to molecular analyses to identify both ECM fungi and host species. While 136 ECM fungal species were identified in total, their communities were significantly different between host groups, confirming the existence of the host effect on ECM fungal communities. None of the 68 ECM fungal species found on P. japonica belonged to Pseudotsuga-specific lineages (e.g., Rhizopogon and Suillus subgroups) that are common in North America. Most of ECM fungi on P. japonica were shared with other host fungi or phylogenetically close to known ECM fungi on other hosts in Asia. These results suggest that after migrating, Pseudotsuga-specific fungal lineages may have become extinct in small isolated populations in Japan. Instead, most of the ECM fungal symbionts on P. japonica likely originated from host shifts in the region.

Wildfire severity in forests is projected to increase with warming and drying conditions associated with climate change. Our objective was to determine the impact of wildfire and clearcutting severity on the ectomycorrhizal fungal (EMF) community of Douglas-fir seedlings in the dry forests of interior British Columbia, Canada. We located our study within and surrounding the area of the McLure fire (August 2003). We hypothesized that disturbance would affect EMF community assembly due to reductions in fungal inoculum. Five treatments representing a range of disturbance severities were compared: high severity burn, low severity burn, screefed clearcut (manual removal of forest floor), clearcut, and undisturbed forest. EMF communities in the undisturbed forest were more complex than those in all disturbance treatments. However, aspects of community assembly varied with disturbance type, where the burn treatments had the simplest communities. After 4 months, regenerating seedlings in the burn treatments had the lowest colonization, but seedlings in all treatments were fully colonized within 1 year. EMF communities were similar among the four disturbance types, largely due to dominance of Wilcoxina throughout the study period. However, forest floor retention influenced community assembly as the EMF in the clearcut treatment, where forest floor was retained, had levels of diversity and richness comparable to the undisturbed forest. Overall, the results suggest that increasing forest floor disturbance can alter EMF community assembly in the first year of regeneration. A correlation between poorly colonized seedlings and seedling productivity also suggests a role for productivity in influencing community assembly.

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.

Douglas-fir (DF) (Pseudotsuga menziesii) is one of the largest and most economically important coniferous species in western North America. Its productivity is greatly affected by the root rot fungus Phellinus sulphurascens Pilát. Evidence of resistance by DF to fungal root pathogens such as P. sulphurascens has been reported but mechanisms of resistance in this compatible pathosystem are not yet known. To better understand the DF-P. sulphurascens interaction, especially at the molecular level, we selected 12 diverse plant genes already identified as defense-related from a cDNA library constructed using root tissues from P. sulphurascens-infected DF seedlings. Using quantitative reverse-transcriptase polymerase chain reaction on infected DF root samples collected at five different time points after inoculation, we found that P. sulphurascens infection significantly elevated expression of the 12 selected genes. In most cases the highest expression level was recorded within 2 to 3 days after inoculation. The constructed cDNA library, which is enriched with defense-related host genes and a number of fungal genes, will continue to serve as a useful resource for future larger-scale gene discovery and functional research on the P. sulphurascens and DF pathosystem.

Understanding ectomycorrhizal fungal (EMF) community structure is limited by a lack of taxonomic resolution and autecological information. Rhizopogon vesiculosus and Rhizopogon vinicolor (Basidiomycota) are morphologically and genetically related species. They are dominant members of interior Douglas-fir (Pseudotsuga menziesii var. glauca) EMF communities, but mechanisms leading to their coexistence are unknown. We investigated the microsite associations and foraging strategy of individual R. vesiculosus and R. vinicolor genets. Mycelia spatial patterns, pervasiveness and root colonization patterns of fungal genets were compared between Rhizopogon species and between xeric and mesic soil moisture regimes. Rhizopogon spp. mycelia were systematically excavated from the soil and identified using microsatellite DNA markers. Rhizopogon vesiculosus mycelia occurred at greater depth, were more spatially pervasive, and colonized more tree roots than R. vinicolor mycelia. Both species were frequently encountered in organic layers and between the interface of organic and mineral horizons. They were particularly abundant within microsites associated with soil moisture retention. The occurrence of R. vesiculosus shifted in the presence of R. vinicolor towards mineral soil horizons, where R. vinicolor was mostly absent. This suggests that competition and foraging strategy may contribute towards the vertical partitioning observed between these species. Rhizopogon vesiculosus and R. vinicolor mycelia systems occurred at greater mean depths and were more pervasive in mesic plots compared with xeric plots. The spatial continuity and number of trees colonized by genets of each species did not significantly differ between soil moisture regimes., (© 2012 Blackwell Publishing Ltd.)

Ectomycorrhizal (EM) networks (MN) are thought to be an important mode of EM fungal colonization of coniferous seedlings. How MNs affect EM communities on seedlings, and how this varies with biotic and abiotic factors, is integral to understanding their importance in seedling establishment. We examined EM fungal community similarity between mature trees and conspecific interior Douglas-fir (Pseudotsuga menziesii var. glauca) seedlings in two experiments where seed and nursery-grown seedlings originating from different locations were planted at various distances from trees along a climatic gradient. At harvest, trees shared 60% of their fungal taxa in common with outplanted seedlings and 77% with germinants, indicating potential for seedlings to join the network of residual trees. In both experiments, community similarity between trees and seedlings increased with drought. However, community similarity was lower among nursery seedlings growing at 2.5 m from trees when they were able to form an MN, suggesting MNs reduced seedling EM fungal richness. For field germinants, MNs resulted in lower community similarity in the driest climates. Distance from trees affected community similarity of nursery seedlings to trees, but there was no interaction of provenance with MNs in their effect on similarity in either nursery seedlings or field germinants as hypothesized. We conclude that MNs of trees influence EM colonization patterns of seedlings, and the strength of these effects increases with climatic drought., (© Springer-Verlag 2012)

Assisted migration of forest trees has been widely proposed as a climate change adaptation strategy, but moving tree populations to match anticipated future climates may disrupt the geographically based, coevolved association suggested to exist between host trees and ectomycorrhizal fungal (EMF) communities. We explored this issue by examining the consistency of EMF communities among populations of 40 year-old Douglas-fir (Pseudotsuga menziesii var. menziesii) trees in a common-garden field trial using four provenances from contrasting coastal climates in southwestern British Columbia. Considerable variation in EMF community composition within test sites was found, ranging from 0.38 to 0.65 in the mean similarity index, and the divergence in EMF communities from local populations increased with site productivity. Clinal patterns in colonization success were detected for generalist and specialist EMF species on only the two productive test sites. Host population effects were limited to EMF species abundance rather than species loss, as richness per site averaged 15.0 among provenances and did not differ by transfer extent (up to 450 km), while Shannon's diversity index declined slightly. Large differences in colonization rates of specialist fungi, such as Tomentella stuposa and Clavulina cristata, raise the possibility that EMF communities maladapted to soil conditions contributed to the inferior growth of some host populations on productive sites. The results of the study suggest locally based specificity in host-fungal communities is likely a contributing factor in the outcome of provenance trials, and should be a consideration in analyzing seed-transfer effects and developing strategies for assisted migration.

Mycorrhizal roots are frequently colonized by fungi of the Phialocephala fortinii s.l.-Acephala applanata species complex (PAC). These ascomycetes are common and widespread colonizers of tree roots. Some PAC strains reduce growth increments of their hosts but are beneficial in protecting roots against pathogens. Nothing is known about the effects of PAC on mycorrhizal fungi and the PAC-mycorrhiza association on plant growth, even though these two fungal groups occur closely together in natural habitats. We expect reduced colonization rates and reduced negative effects of PAC on host plants if roots are co-colonized by an ectomycorrhizal fungus (ECM). Depending on the temperature regime interactions among the partners in this tripartite ECM-PAC-plant system might also change. To test our hypotheses, effects of four PAC genotypes (two pathogenic and two non-pathogenic on the Norway spruce), mycorrhization by Laccaria bicolor (strain S238N) and two temperature regimes (19°C and 25°C) on the biomass of the Douglas-fir (Pseudotsuga menziesii) and Norway spruce (Picea abies) seedlings were studied. Mycorrhization compensated the adverse effects of PAC on the growth of the Norway spruce at both temperatures. The growth of the Douglas-fir was not influenced either by PAC or mycorrhization at 19°C, but at 25°C mycorrhization had a similar protective effect as in the Norway spruce. The compensatory effects probably rely on the reduction of the PAC-colonization density by mycorrhizae. Temperature and the PAC strain only had a differential effect on the biomass of the Norway spruce but not on the Douglas-fir. Higher temperature reduced mycorrhization of both hosts. We conclude that ectomycorrhizae form physical and/or physiological barriers against PAC leading to reduced PAC-colonization of the roots. Additionally, our results indicate that global warming could cause a general decrease of mycorrhization making primary roots more accessible to other symbionts and pathogens.

Ectomycorrhizal fungi (EMF) provide nutrients to their hosts by means of hyphae that extend beyond nutrient-depleted rhizosphere soil. Soil bacteria may compete with EMF for nutrients or may act synergistically to enhance nutrient supply to hosts. To assess the interactions between hyphae and bacteria, two types of small, sand-filled mesh bags were incubated in a Pseudotsuga menziesii/Betula papyrifera forest. The bags allowed ingrowth by EMF (35-μm mesh) or excluded hyphae (0.5-μm mesh), while allowing migration of soil bacteria. After incubation, bacteria were isolated from bags using a method to enrich for Gram-positive bacteria. Isolates were assayed for phosphatase and N-acetyl glucosaminidase (NAGase) activities to assess the potential to access organic phosphorus and nitrogen. The average phosphatase activities were higher in exclusion than ingrowth bags, while NAGase activities did not differ. Streptomyces isolates, which are expected to be strong competitors and antagonists of EMF, were more prevalent in ingrowth bags and yet had lower phosphatase activities. Furthermore, there were no indications of antagonism between fungi and Streptomyces, as there were no increases in NAGase activities in ingrowth bags. We conclude that fungal hyphae can structure components of the soil bacterial community for decreased extracellular enzyme production., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Phaeocryptopus gaeumannii, the cause of Swiss needle-cast, is widely distributed in plantations of Douglas-fir in many parts of the world. Nevertheless, information remains limited on its precise effect on stand growth, particularly in relation to regional climate, and on its consequent economic cost. In New Zealand, the spread of P. gaeumannii over a period of ≈30 years following its discovery in 1959 was closely monitored, and the timing of its arrival in different forests is known. This information was coupled with data from permanent sample plots in order to quantify the associated historical growth increment loss. Analyses revealed a steady decline in growth rate over the period from the first appearance of P. gaeumannii to a point when it stabilized at a lower increment level 14 to 20 years later. The cumulative mean reduction was 25% for mean top height, 27% for basal area, and 32% for stem volume. Volume growth rate decline was greater in the North Island (35%) than the South Island (23%) of New Zealand. These reductions in volume growth are estimated to equate to a loss in net present value of $NZ2,620 ha(-1) and $NZ1,470 ha(-1) for the North and South Islands, respectively, using a discount rate of 6%. Mortality did not increase as a result of infection by P. gaeumannii. The disease had less effect on cooler sites, especially those with low spring minimum temperatures (P < 0.001). Negligible growth decline occurred on sites with daily minimum October temperatures averaging <3.2°C.

The abundance of nifH, nirS, and nirK gene fragments involved in nitrogen (N) fixation and denitrification in thinned second-growth Douglas-fir (Pseudotsuga menziesii subsp. menziesii [Mirb.] Franco) forest soil was investigated by using quantitative real-time PCR. Prokaryotic N cycling is an important aspect of N availability in forest soil. The abundance of universal nifH, Azotobacter sp.-specific nifH (nifH-g1), nirS, and nirK gene fragments in unthinned control and 30, 90, and 100% thinning treatments were compared at two long-term research sites on Vancouver Island, Canada. The soil was analyzed for organic matter (OM), total carbon (C), total N, NH₄-N, NO₃-N, and phosphorus (P). The soil horizon accounted for the greatest variation in nutrient status, followed by the site location. The 30% thinning treatment was associated with significantly greater nifH-g1 abundance than the control treatment in one site; at the same site, nirS in the mineral soil horizon was significantly reduced by thinning. The abundance of nirS genes significantly correlated with the abundance of nirK genes. In addition, significant correlations were observed between nifH-g1 abundance and C and N in the organic horizon and between nirS and nirK and N in the mineral horizon. Overall, no clear influence of tree thinning on nifH, nirS, and nirK was observed. However, soil OM, C, and N were found to significantly influence N-cycling gene abundance.

Communities of archaea, bacteria, and fungi were examined in forest soils located in the Oregon Coast Range and the inland Cascade Mountains. Soils from replicated plots of Douglas-fir (Pseudotsuga menziesii) and red alder (Alnus rubra) were characterized using fungal ITS (internal transcribed spacer region), eubacterial 16S rRNA, and archaeal 16S rRNA primers. Population size was measured with quantitative (Q)-PCR and composition was examined using length heterogeneity (LH)-PCR for fungal composition, terminal restriction fragment length (T-RFLP) profiles for bacterial and archaeal composition, and sequencing to identify dominant community members. Whereas fungal and archaeal composition varied between sites and dominant tree species, bacterial communities only varied between sites. The abundance of archaeal gene copy numbers was found to be greater in coastal compared to montane soils accounting for 11% of the prokaryotic community. Crenarchaea groups 1.1a-associated, 1.1b, 1.1c, and 1.1c-associated were putatively identified. A greater abundance of Crenarchaea 1.1b indicator fragments was found in acidic (pH 4) soils with low C:N ratios under red alder. In coastal soils, 25% of fungal sequences were putatively identified as basidiomycetous yeasts belonging to the genus Cryptococcus. Although the function of these yeasts in soil is not known, they could significantly contribute to decomposition processes in coastal soils distinguished by rapid tree growth, high N content, low pH, and frequent water-saturation events.

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.

Laminated root rot (LRR) disease, caused by the fungus Phellinus sulphurascens, is a major threat to coastal Douglas-fir (DF) (Pseudotsuga menziesii) forests in western North America. Understanding host-pathogen interactions of this pathosystem is essential to manage this important conifer root disease. Our research objectives were to identify DF pathogenesis-related (PR) genes and analyze their expression patterns over the course of infection. We constructed a cDNA library of Phellinus sulphurascens-infected DF seedling roots and sequenced a total of 3,600 random cDNA clones from this library. One of the largest groups of identified genes (203 cDNA clones) matched with chitinase genes reported in other plant species. We identified at least three class II and six class IV chitinase genes from DF seedlings. Quantitative reverse-transcriptase polymerase chain reaction analyses showed significant differential expression patterns locally in root tissues and systemically in needle tissues after fungal invasion. Nonetheless, there was a common trend in gene expression patterns for most of the chitinase genes: an upregulation within 12 h of pathogen inoculation followed by down-regulation within 2 to 3 days postinoculation (dpi), and then further upregulation within 5 to 7 dpi. Western immunoblot data showed differential accumulation of class IV chitinases in Phellinus sulphurascens-infected DF seedlings. Further detailed functional analyses will help us to understand the specific role of DF chitinases in defense against Phellinus sulphurascens infection.

A quantitative polymerase chain reaction assay was developed that could detect DNA of Rhabdocline pseudotsugae and R. oblonga among DNA of Douglas fir needles to a limit as low as three copies of target DNA. Differential infection rates of two varieties (seed sources) of Douglas fir interplanted in a field were studied in relation to staggered bud breaks. Infection of Douglas fir var. San Isabel corresponded to ascospore release times for Rhabdocline spp., whereas infection of var. Shuswap Lake did not occur throughout the spore release period during 2 years of study, despite abundant inoculum and adequate moisture during bud break. Rhabdocline spp. DNA was never detected in Shuswap Lake and disease symptoms were not observed in any year. We provide evidence that Shuswap Lake is resistant and probably immune to Rhabdocline spp. infection and Rhabdocline needlecast under Michigan conditions.

Commercial nursery practices usually fail to promote mycorrhization of interior Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco var. glauca (Beissn.) Franco] seedlings in British Columbia, which may account for their poor performance following planting in the field. We tested the effects of four nursery cultivation factors (nitrogen fertilization, phosphorus fertilization, watering, and soil aeration) and field soil addition on mycorrhization, survival, growth, and biomass allocation of interior Douglas-fir seedlings in a series of greenhouse experiments. Where field soil was added to the growing medium, mycorrhization and root/shoot ratios were maximized at lower levels of mineral nutrient application and aeration. Where field soil was not added, mycorrhization was negligible across all fertilization and aeration treatments, but root/shoot ratio was maximized at lower levels of mineral nutrients and the highest level of aeration. Regardless of whether field soil was added, intermediate levels of soil water resulted in the best mycorrhizal colonization and root/shoot ratios. However, field soil addition reduced seedling mortality at the two lowest water levels. A cluster analysis placed ectomycorrhizal morphotypes into three groups (Mycelium radicis-atrovirens Melin, Wilcoxina, and mixed) based on their treatment response, with all but two morphotypes in the mixed group whose abundance was maximized under conditions common to advanced seedling establishment. For maximal mycorrhization and root development of interior Douglas-fir seedlings, nurseries should minimize addition of nitrogen and phosphorus nutrients, maximize aeration, provide water at moderate rates, and, where possible, add small amounts of field soil to the growing medium.

Here, the genome sequence of the ectomycorrhizal basidiomycete Laccaria bicolorwas explored with the aim of constructing a genome-wide inventory of genes involved in fatty acid metabolism. Sixty-three genes of the major pathways were annotated and validated by the detection of the corresponding transcripts. Seventy-one per cent belonged to multigene families of up to five members. In the mycelium of L. bicolor, 19 different fatty acids were detected, including at low concentrations palmitvaccenic acid (16:1(11Z)), which is known to be a marker for arbuscular mycorrhizal fungi. The pathways of fatty acid biosynthesis and degradation in L. bicolor were reconstructed using lipid composition, gene annotation and transcriptional analysis. Annotation results indicated that saturated fatty acids were degraded in mitochondria, whereas degradation of modified fatty acids was confined to peroxisomes. Fatty acid synthase (FAS) was the second largest protein annotated in L. bicolor. Phylogenetic analysis indicated that L. bicolor, Ustilago maydis and Coprinopsis cinerea have a vertebrate-like type I FAS encoded as a single protein, whereas in other basidiomycetes, including the human pathogenic basidiomycete Cryptococcus neoformans, and in most ascomycetes FAS is composed of the two structurally distinct subunits α and β.

Interactions between roots of Douglas-fir (DF; Pseudotsuga menziesii) seedlings and the laminated root rot fungus Phellinus sulphurascens were investigated using scanning and transmission electron microscopy and immunogold labelling techniques. Scanning electron micrographs revealed that P. sulphurascens hyphae colonize root surfaces and initiate the penetration of root epidermal tissues by developing appressoria within 2 d postinoculation (dpi). During early colonization, intra- and intercellular fungal hyphae were detected. They efficiently disintegrate cellular components of the host including cell walls and membranes. P. sulphurascens hyphae penetrate host cell walls by forming narrow hyphal tips and a variety of haustoria-like structures which may play important roles in pathogenic interactions. Ovomucoid-WGA (wheat germ agglutinin) conjugated gold particles (10 nm) confirmed the occurrence and location of P. sulphurascens hyphae, while four specific host pathogenesis-related (PR) protein antibodies conjugated with protein A-gold complex (20 nm) showed the localization and abundance of these PR proteins in infected root tissues. A thaumatin-like protein and an endochitinase-like protein were both strongly evident and localized in host cell membranes. A DF-PR10 protein was localized in the cell walls and cytoplasm of host cells while an antimicrobial peptide occurred in host cell walls. A close association of some PR proteins with P. sulphurascens hyphae suggests their potential antifungal activities in DF roots.

In this study, basidiomycete isolates that possessed a strong ability to degrade chromated copper arsenate (CCA)-treated wood were characterized. These fungal isolates, which were collected from CCA-treated pine log wastes, showed no recognizable morphological properties on culture media. Nucleotide sequence analysis of the large subunit rDNA of the isolates revealed that they were one species. Based on the high sequence similarity (>95%) and close phylogenetic relationship with several known species of Crustoderma, the fungal isolates characterized in this study were classified as a Crustoderma sp. In a wood degradation test, Crustoderma isolate KUC8611 produced a remarkably higher weight loss in CCA-treated Pinus radiata (68.7%), Pseudotsuga menziesii (39.7%), and Tsuga heterophylla (38.5%) wood than other evaluated basidiomycete species, including Crustoderma flavescens and Crustoderma corneum. In addition, extracellular enzymes for cellulose and protein degradation were detected when the isolates were cultured in chromogenic media, which supports the finding that isolate KUC8611 is a wood degrader. Furthermore, an in vitro test for metal tolerance revealed that isolate KUC8611 showed strong arsenic tolerance, but that it could not tolerate copper. Finally, isolate KUC8611 produced lower amounts of oxalic acid than copper-tolerant fungi such as Fomitopsis palustris and Antrodia vaillantii. To the best of our knowledge, this is the first study to report the degradation of CCA-treated wood by a Crustoderma species.

The distribution of dry Douglas-fir forests in western North America is expected to shift northward with climate change and disappear from the grassland interface in the southern interior of British Columbia. This shift may be accentuated by clearcutting, a common harvesting practice that aims to reduce the competitive effects of residual mature trees on new regeneration, but in so doing, ignores their facilitative effects. In this study, we investigated the net effects of competition from and facilitation by mature trees retained on harvested sites on seedling establishment in the dry interface Douglas-fir forests. We demonstrate that access to a mycorrhizal network (MN) and proximity to trees have important influences on seedling performance. On six sites, we established trenched plots around 24 mature Pseudotsuga menziesii var. glauca (Douglas-fir) trees, then planted Douglas-fir seedlings into four mesh treatments that served to restrict MN access (i.e., planted into mesh bags with 0.5-, 35-, or 250-microm pores, or without mesh) or into impermeable bags (grown in isolation) at four distances (0.5, 1.0, 2.5, or 5.0 m). Seedling survival tended to be greater and water stress lower where seedlings had full access to the MN. Seedling height, shoot biomass, needle biomass, and nutrient uptake peaked at 2.5-5.0 m from mature trees. Seedlings 0.5 m from mature trees had lower CO2 assimilation rates and wood delta(13)C compared to seedlings 5.0 m away. Competition for soil resources was highest near mature trees but facilitation was relatively greater at further distances, resulting in a zone of net benefit for seedlings. These results show that intraspecific tree-seedling interactions are both competitive and facilitative in dry Douglas-fir forests, and that they are spatially dependent. After disturbance, maintaining residual mature trees may be important for their beneficial regeneration zones.

This study determined nitrification activity and nitrifier community composition in soils under stands of red alder (Alnus rubra) and Douglas fir (Pseudotsuga menziesii) at two sites in Oregon. The H.J. Andrews Experimental Forest, located in the Cascade Mountains of Oregon, has low net N mineralization and gross nitrification rates. Cascade Head Experimental Forest, in the Coast Range, has higher net N mineralization and nitrification rates and soil pH is lower. Communities of putative bacterial [ammonia-oxidizing bacteria (AOB)] and archaeal [ammonia-oxidizing archaea (AOA)] ammonia oxidizers were examined by targeting the gene amoA, which codes for subunit A of ammonia monooxygenase. Nitrification potential was significantly higher in red alder compared with Douglas-fir soil and greater at Cascade Head than H.J. Andrews. Ammonia-oxidizing bacteria amoA genes were amplified from all soils, but AOA amoA genes could only be amplified at Cascade Head. Gene copy numbers of AOB and AOA amoA were similar at Cascade Head regardless of tree type (2.3-6.0 x 10(6)amoA gene copies g(-1) of soil). DNA sequences of amoA revealed that AOB were members of Nitrosospira clusters 1, 2 and 4. Ammonia-oxidizing bacteria community composition, determined by terminal restriction fragment length polymorphism (T-RFLP) profiles, varied among sites and between tree types. Many of the AOA amoA sequences clustered with environmental clones previously obtained from soil; however, several sequences were more similar to clones previously recovered from marine and estuarine sediments. As with AOB, the AOA community composition differed between red alder and Douglas-fir soils.

We studied the effect of ectomycorrhizal fungi on bacterial communities colonizing roots of Douglas fir (Pseudotsuga menziesii). Mycorrhizal tips were cleaned of soil and separated based on gross morphological characteristics. Sequencing of the internal transcribed spacers of the nuclear rRNA gene cluster indicated that the majority of the tips were colonized by fungi in the Russulaceae, with the genera Russula and Lactarius comprising 70% of the tips. Because coamplification of organellar 16S rRNA genes can interfere with bacterial community analysis of root tips, we developed and tested a new primer pair that permits amplification of bacterial 16S rRNA genes but discriminates more effectively against organellar sequences than commonly used bacterial primer sets. We then used terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis of the 16S rRNA gene to examine differences in bacterial communities associated with the mycorrhizal tips. Cluster analysis of T-RFLP profiles indicated that there were different bacterial communities among the root tips; however, the communities did not seem to be affected by the taxonomic identity of the ectomycorrhizal fungi. Terminal restriction fragment profiling and sequencing of cloned partial 16S rRNA genes indicated that most bacteria on the ectomycorrhizal tips were related to the Alphaproteobacteria and the Bacteroidetes group.

Acidification of forest ecosystems leads to increased plant availability of the micronutrient manganese (Mn), which is toxic when taken up in excess. To investigate whether ectomycorrhizas protect against excessive Mn by improving plant growth and nutrition or by retention of excess Mn in the hyphal mantle, seedlings of two populations of Douglas fir (Pseudotsuga menziesii), two varieties, one being menziesii (DFM) and the other being glauca (DFG), were inoculated with the ectomycorrhizal fungus Rhizopogon subareolatus in sand cultures. Five months after inoculation, half of the inoculated and non-inoculated seedlings were exposed to excess Mn in the nutrient solution for further 5 months. At the end of this period, plant productivity, nutrient concentrations, Mn uptake and subcellular compartmentalisation were evaluated. Non-inoculated, non-stressed DFM plants produced about 2.5 times more biomass than similarly treated DFG. Excess Mn in the nutrient solution led to high accumulation of Mn in needles and roots but only to marginal loss in biomass. Colonisation with R. subareolatus slightly suppressed DFM growth but strongly reduced that of DFG (-50%) despite positive effects of mycorrhizas on plant phosphorus nutrition. Growth reductions of inoculated Douglas fir seedlings were unexpected since the degree of mycorrhization was not high, i.e. ca. 30% in DFM and 8% in DFG. Accumulation of high Mn was not prevented in inoculated seedlings. The hyphal mantle of mycorrhizal root tips accumulated divalent cations such as Ca, but not Mn, thus not providing a barrier against excessive Mn uptake into the plants associated with R. subareolatus.

The colonization of leaf litter by saprobic fungi was studied in old-growth and post-harvest successional Douglas-fir forests on southeast Vancouver Island, British Columbia. This study focused on leaf litter of salal (Gaultheria shallon Pursh.), a dominant understory shrub in all stands. Salal litter is characterized by the occurrence of bleached portions attributable to fungal colonization of the litter and to the variable decomposition of recalcitrant compounds, such as lignin. Analyses of proximate chemical fractions, fungal assemblages on the bleached leaf area, and pure culture decomposition assays indicated that Marasmius sp. and Coccomyces sp. were responsible for rapid decomposition and bleaching of salal leaf litter. The bleached area accounted for 17%-22% of total area of salal leaf litter collected in immature (40-60 years old), mature (85-105 years old), and old-growth (more than 290 years old) stands, but for only 2% in regeneration (5-15 years old) stands. The reduction of bleached leaf area occupied by Marasmius sp. and Coccomyces sp. in regeneration stands could be due to the changes in microenvironmental conditions on the forest floor, in litter quality, or in food-web structure in soils. The decrease of fungi able to decay recalcitrant compounds may lead to a reduction of salal decomposition rates in clear-cut sites that would persist until canopy closure occurs.

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.