Growth on Douglas fir media facilitates Cryptococcus virulence factor production and enhances fungal survival against environmental and immune stressors.

The yeasts Cryptococcus neoformans and Cryptococcus gattii are fungal pathogens that can be isolated from the environment, including the surfaces of many plants. Cryptococcus gattii caused an outbreak on Vancouver Island, British Columbia beginning in 1999 that has since spread to the Pacific Northwest of the United States. Coastal Douglas fir (Pseudotsuga menziesii) is an important lumber species and a major component of the ecosystems in this area. Previous research has explored Cryptococcus survival and mating on Douglas fir plants and plant-derived material, but no studies have been done on the production of cryptococcal virulence factors by cells grown on those media. Here, we investigated the effects of growth on Douglas fir-derived media on the production of the polysaccharide capsule and melanin, two of the most important cryptococcal virulence factors. We found that while the capsule was mostly unchanged by growth in Douglas fir media compared to cells grown in defined minimal media, Cryptococcus spp. can use substrates present in Douglas fir to synthesize functional and protective melanin. These results suggest mechanisms by which Cryptococcus species may survive in the environment and emphasize the need to explore how association with Douglas fir trees could affect its epidemiology for human cryptococcosis.

Cryptococcus gattii is a fungal pathogen that can be found in the environment. It is responsible for causing an outbreak in British Columbia, Canada, in the late 90s. In our study, we created media from Douglas fir, a tree commonly found in the affected areas. We examined the production of virulence factors by Cryptococcus cells grown in this media.

Genetic architecture of disease resistance and tolerance in Douglas-fir trees.

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.

Identifying potential provenances for climate-change adaptation using spatially variable coefficient models.

BACKGROUND: Selection of climate-change adapted ecotypes of commercially valuable species to date relies on DNA-assisted screening followed by growth trials. For trees, such trials can take decades, hence any approach that supports focussing on a likely set of candidates may save time and money. We use a non-stationary statistical analysis with spatially varying coefficients to identify ecotypes that indicate first regions of similarly adapted varieties of Douglas-fir (Pseudotsuga menziesii (Mirbel) Franco) in North America. For over 70,000 plot-level presence-absences, spatial differences in the survival response to climatic conditions are identified. RESULTS: The spatially-variable coefficient model fits the data substantially better than a stationary, i.e. constant-effect analysis (as measured by AIC to account for differences in model complexity). Also, clustering the model terms identifies several potential ecotypes that could not be derived from clustering climatic conditions itself. Comparing these six identified ecotypes to known genetically diverging regions shows some congruence, as well as some mismatches. However, comparing ecotypes among each other, we find clear differences in their climate niches. CONCLUSION: While our approach is data-demanding and computationally expensive, with the increasing availability of data on species distributions this may be a useful first screening step during the search for climate-change adapted varieties. With our unsupervised learning approach being explorative, finely resolved genotypic data would be helpful to improve its quantitative validation.

Niche dimensions in soil oribatid mite community assembly under native and introduced tree species.

Forest soils are a critical component of terrestrial ecosystems and host a large number of animal decomposer species. One diverse and abundant decomposer taxon is oribatid mites (Acari: Oribatida), whose species composition varies with forest type and tree species composition. We used functional traits that indicate different niche dimensions, to infer assembly processes of oribatid mite communities in monocultures and mixed forests of native and introduced tree species. We found that coexisting species differed more in the resource-related niche dimension, i.e., reproductive mode and trophic guild, than in the morphological dimension, e.g., body length and width, sclerotization and concealability. These results suggest that both filtering and partitioning processes structure oribatid mite communities. In native European beech forests, but not in non-native Douglas fir forests, oribatid mites were mainly structured by filtering processes acting via traits related both to environmental tolerance and to resources. Furthermore, oribatid mite trait diversity, but not phylogenetic diversity, differed significantly between monocultures and mixed forests, demonstrating that multidimensional diversity indices provide additional information on soil biodiversity. Overall, the study provides evidence that traits representing different niche dimensions need to be considered for understanding assembly processes in soil animal communities and thereby soil biodiversity.

Weak local adaptation to drought in seedlings of a widespread conifer.

Tree seedlings from populations native to drier regions are often assumed to be more drought tolerant than those from wetter provenances. However, intraspecific variation in drought tolerance has not been well-characterized despite being critical for developing climate change mitigation and adaptation strategies, and for predicting the effects of drought on forests. We used a large-scale common garden drought-to-death experiment to assess range-wide variation in drought tolerance, measured by decline of photosynthetic efficiency, growth, and plastic responses to extreme summer drought in seedlings of 73 natural populations of the two main varieties of Douglas-fir (Pseudotsuga menziesii var. menziesii and var. glauca). Local adaptation to drought was weak in var. glauca and nearly absent in menziesii. Var. glauca showed higher tolerance to drought but slower growth than var. menziesii. Clinal variation in drought tolerance and growth species-wide was mainly associated with temperature rather than precipitation. A higher degree of plasticity for growth was observed in var. menziesii in response to extreme drought. Genetic variation for drought tolerance in seedlings within varieties is maintained primarily within populations. Selective breeding within populations may facilitate adaptation to drought more than assisted gene flow.

Genome sequences of three genetic lineages of the fungus Nothophaeocryptopus gaeumannii, the causal agent of Swiss needle cast on Douglas-fir trees.

Here, we present the nearly complete genome sequences of the three main genetic lineages of Nothophaeocryptopus gaeumannii, an endophytic ascomycete fungus responsible for Swiss needle cast, a foliar disease that is emerging as a significant threat to the Douglas-fir tree in its natural distribution range.

Uncovering epigenetic and transcriptional regulation of growth in Douglas-fir: identification of differential methylation regions in mega-sized introns.

Tree growth performance can be partly explained by genetics, while a large proportion of growth variation is thought to be controlled by environmental factors. However, to what extent DNA methylation, a stable epigenetic modification, contributes to phenotypic plasticity in the growth performance of long-lived trees remains unclear. In this study, a comparative analysis of targeted DNA genotyping, DNA methylation and mRNAseq profiling for needles of 44-year-old Douglas-fir trees (Pseudotsuga menziesii (Mirb.) Franco) having contrasting growth characteristics was performed. In total, we identified 195 differentially expressed genes (DEGs) and 115 differentially methylated loci (DML) that are associated with genes involved in fitness-related processes such as growth, stress management, plant development and energy resources. Interestingly, all four intronic DML were identified in mega-sized (between 100 and 180 kbp in length) and highly expressed genes, suggesting specialized regulation mechanisms of these long intron genes in gymnosperms. DNA repetitive sequences mainly comprising long-terminal repeats of retroelements are involved in growth-associated DNA methylation regulation (both hyper- and hypomethylation) of 99 DML (86.1% of total DML). Furthermore, nearly 14% of the DML was not tagged by single nucleotide polymorphisms, suggesting a unique contribution of the epigenetic variation in tree growth.

Transcriptomic monitoring of Douglas-fir heartwood formation.

OBJECTIVES: Molecular cues linked to heartwood formation open new (complementary) perspectives to genetic breeding programs of Douglas-fir, a tree species largely cultivated in Europe for the natural durability and civil engineering properties of its wood. DATA DESCRIPTION: RNAs from a single genotype of Douglas-fir, extracted from three distinct wood zones (outer sapwood, inner sapwood and transition zone) at four vegetative seasons to generate an extensive RNA-seq dataset used to apprehend the in-wood dynamic and seasonality of heartwood formation in this hardwood model species. Previously published data collected on somatic embryos of the same genotype could be merged with the present dataset to upgrade grade the Douglas-fir reference transcriptome.

Tree-ring δ15N as an indicator of nitrogen dynamics in stands with N2-fixing Alnus rubra.

Tree-ring δ15N may depict site-specific, long-term patterns in nitrogen (N) dynamics under N2-fixing species, but field trials with N2-fixing tree species are lacking and the relationship of temporal patterns in tree-ring δ15N to soil N dynamics is controversial. We examined whether the tree-ring δ15N of N2-fixing red alder (Alnus rubra Bong.) would mirror N accretion rates and δ15N of soils and whether the influence of alder-fixed N could be observed in the wood of a neighboring conifer. We sampled a 27-year-old replacement series trial on south-eastern Vancouver Island, with red alder and coastal Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) planted in five proportions (0/100, 11/89, 25/75, 50/50 and 100/0) at a uniform stem density. An escalation in forest floor N content was evident with an increasing proportion of red alder, equivalent to a difference of ~750 kg N ha-1 between 100% Douglas-fir versus 100% alder. The forest floor horizon also had high δ15N values in treatments with more red alder. Red alder had a consistent quadratic fit in tree-ring δ15N over time, with a net increase of $\sim$1.5‰, on average, from initial values, followed by a plateau or slight decline. Douglas-fir tree-ring δ15N, in contrast, was largely unchanged over time (in three of four plots) but was significantly higher in the 50/50 mix. The minor differences in current leaf litter N content and δ15N between alder and Douglas-fir, coupled with declining growth in red alder, suggests the plateau or declining trend in alder tree-ring δ15N could coincide with lower N2-fixation rates, potentially by loss in alder vigor at canopy closure, or down-regulation via nitrate availability.

Douglas-fir plantations impact stream and groundwater chemistry in western Europe: Insights from three case studies in France and Luxembourg.

In rural areas, nitrate concentrations in surface waters most often originate from the leaching of excess N fertilizer in agricultural lands, whereas forested catchments often have good water quality. However, Douglas-fir plantations may induce nitrogen cycle unbalances which may lead to an excess of nitrate production in the soil. We hypothesize that the excess of production of nitrate in the soil and nitrate leaching to streamwater is greater in catchments planted with Douglas fir. We used paired catchments in both France and Luxembourg with different land covers (Douglas-fir, Spruce, Deciduous, Grassland and clearcut) which were monitored over a 3-5 year period in order to assess the effect of Douglas-fir plantations on the chemical composition of surface water. Nitrate concentration in the soil and groundwater were also monitored. The results show that nitrate concentrations in streams draining Douglas-fir catchments were two to ten times higher than in streams draining other land covers, but were similar to the clearcut catchment. Nitrate concentrations under Douglas-fir in groundwater (up to 50 mg L-1) and in the soil were also higher than under all other land covers. Soil nitrate concentration was related to stream nitrate concentration. This suggests that soil processes, through excessive nitrate production under Douglas-fir, are driving the nitrate concentration in the stream water and our hypothesis of a transfer of a fairly large proportion of this excessive production from the soil to the stream is supported. This study also shows that nitrate concentrations in surface and ground waters in rural areas could also originate from Douglas fir forested catchments. The impact of Douglas-fir is nevertheless reduced downstream through a dilution effect: mixing tree species at the catchment scale could thus be a solution to mitigate the effect of Douglas-fir on nitrate concentration in surface waters.

Differences in heat tolerance, water use efficiency and growth among Douglas-fir families and varieties evidenced by GWAS and common garden studies.

Severe and frequent heat and drought events challenge the survival and development of long-generation trees. In this study, we investigated the genomic basis of heat tolerance, water use efficiency and growth by performing genome-wide association studies in coastal Douglas-fir (Pseudotsuga menziesii) and intervarietal (menziesii × glauca) hybrid seedlings. GWAS results identified 32 candidate genes involved in primary and secondary metabolism, abiotic stress and signaling, among other functions. Water use efficiency (inferred from carbon isotope discrimination), photosynthetic capacity (inferred from %N), height and heat tolerance (inferred from electrolyte leakage in a heat stress experiment) were significantly different among Douglas-fir families and varieties. High-elevation seed sources had increased water use efficiency, which could be a result of higher photosynthetic capacity. Similarly, families with greater heat tolerance also had higher water use efficiency and slower growth, suggesting a conservative growth strategy. Intervarietal hybrids showed increased heat tolerance (lower electrolyte leakage at 50 and 55 °C) and higher water use efficiency compared with coastal families, suggesting that hybridization might be a source of pre-adapted alleles to warming climates and should be considered for large-scale reforestation projects under increasingly arid conditions.

Host population effects on ectomycorrhizal fungi vary between low and high phosphorus soils of temperate rainforests.

Geographic distinctions in the affinity of tree populations for select ectomycorrhizal fungi (EMF) may occur where strong edaphic pressures act on fungal communities and their hosts. We examine this premise for Pseudotsuga menziesii var. menziesii of southwest British Columbia, using ten native seedlots collected from a range of mean annual precipitation (MAP), as a proxy for podzolization extent and phosphorus (P) deficiencies, and evaluated in contrasting low P and high P soils. After two growing seasons, seedling biomass in the high P soil dwarfed that of the low P soil, and better growth rates under high P were detected for populations from very dry and very wet origins. EMF communities on the high P soil displayed more symmetry among host populations than the low P soil (average community dissimilarity of 0.20% vs. 0.39%, respectively). Seedling foliar P% differed slightly but significantly in relation to MAP of origin. EMF species richness varied significantly among host populations but independently of climatic parameters. There were significant shifts in EMF species abundance related to seedlot MAP, particularly on the low P soil where nonlinear relationships were found for Wilcoxina mikolae, Hyaloscypha finlandica, and Rhizopogon villosulus. Despite efforts to enhance colonization by native fungi, the predominance of ruderal EMF species hindered a realistic evaluation of local adaptation among host-fungi populations. Nevertheless, the shifting affinity in taxa abundance and wider community disparity on low P soil reflected the potential for a consequential host genetic effect related to geographical patterns in P availability across temperate rainforests.

Gymnosperms of Idaho: Chemical Compositions and Enantiomeric Distributions of Essential Oils of Abies lasiocarpa, Picea engelmannii, Pinus contorta, Pseudotsuga menziesii, and Thuja plicata.

Conifers are of great economic value in terms of lumber production, important for construction and other uses such as pulp and paper. They are also important sources of essential oils. Conifer species have been vital to the ethnobotany and traditional herbal medicine of many different Native American groups. The objective of this work was to obtain and analyze the essential oils of several conifer species (Abies lasiocarpa, Picea engelmannii, Pinus contorta, Pseudotsuga menziesii, and Thuja plicata) growing in Idaho. The foliar essential oils were obtained by hydrodistillation and then analyzed by gas chromatographic methods, including GC-MS, GC-FID, and chiral GC-MS. The essential oils were obtained in varying yields from 0.66% up to 4.70%. The essential oil compositions were largely dominated by monoterpene hydrocarbons and oxygenated monoterpenoids. The chiral monoterpenoids were generally rich in the (-)-enantiomers for members of the Pinaceae, but the (+)-enantiomers predominated in the Cupressaceae. The essential oil compositions obtained in this work are qualitatively similar, but quantitatively different, to previously reported compositions and confirm and complement the previous reports. However, this is the first comprehensive analysis of the chiral terpenoid components in these conifer species. Additional research on essential oils of the Pinaceae and Cupressaceae is needed to describe the chemical profiles, chemical compositions, and enantiomeric distributions more reliably in the various species and infraspecific taxa of these two families.

A long-read and short-read transcriptomics approach provides the first high-quality reference transcriptome and genome annotation for Pseudotsuga menziesii (Douglas-fir).

Douglas-fir (Pseudotsuga menziesii) is native to western North America. It grows in a wide range of environmental conditions and is an important timber tree. Although there are several studies on the gene expression responses of Douglas-fir to abiotic cues, the absence of high-quality transcriptome and genome data is a barrier to further investigation. Like for most conifers, the available transcriptome and genome reference dataset for Douglas-fir remains fragmented and requires refinement. We aimed to generate a highly accurate, and complete reference transcriptome and genome annotation. We deep-sequenced the transcriptome of Douglas-fir needles from seedlings that were grown under nonstress control conditions or a combination of heat and drought stress conditions using long-read (LR) and short-read (SR) sequencing platforms. We used 2 computational approaches, namely de novo and genome-guided LR transcriptome assembly. Using the LR de novo assembly, we identified 1.3X more high-quality transcripts, 1.85X more "complete" genes, and 2.7X more functionally annotated genes compared to the genome-guided assembly approach. We predicted 666 long noncoding RNAs and 12,778 unique protein-coding transcripts including 2,016 putative transcription factors. We leveraged the LR de novo assembled transcriptome with paired-end SR and a published single-end SR transcriptome to generate an improved genome annotation. This was conducted with BRAKER2 and refined based on functional annotation, repetitive content, and transcriptome alignment. This high-quality genome annotation has 51,419 unique gene models derived from 322,631 initial predictions. Overall, our informatics approach provides a new reference Douglas-fir transcriptome assembly and genome annotation with considerably improved completeness and functional annotation.

The contribution of Indigenous stewardship to an historical mixed-severity fire regime in British Columbia, Canada.

Indigenous land stewardship and mixed-severity fire regimes both promote landscape heterogeneity, and the relationship between them is an emerging area of research. In our study, we reconstructed the historical fire regime of Ne Sextsine, a 5900-ha dry, Douglas fir-dominated forest in the traditional territory of the T'exelc (Williams Lake First Nation) in British Columbia, Canada. Between 1550 and 1982 CE, we found median fire intervals of 18 years at the plot level and 4 years at the study-site level. Ne Sextsine was characterized by an historical mixed-severity fire regime, dominated by frequent, low-severity fires as indicated by fire scars, with infrequent, mixed-severity fires indicated by cohorts. Differentiating low- from mixed-severity plots over time was key to understanding the drivers of the fire regime at Ne Sextsine. Low-severity plots were coincident with areas of highest use by the T'exelc, including winter village sites, summer fishing camps, and travel corridors. The high fire frequency in low-severity plots ceased in the 1870s, following the smallpox epidemic, the forced relocation of Indigenous peoples into small reserves, and the prohibition of Indigenous burning. In contrast, the mixed-severity plots were coincident with areas where forest resources, such as deer or certain berry species, were important. The high fire frequency in the mixed-severity plots continued until the 1920s when industrial-scale grazing and logging began, facilitated by the establishment of a nearby railway. T'exelc oral histories and archeological evidence at Ne Sextsine speak to varied land stewardship, reflected in the spatiotemporal complexity of low- and mixed-severity fire plots. Across Ne Sextsine, 63% of cohorts established and persisted in the absence of fire after colonial impacts beginning in the 1860s, resulting in a dense, homogeneous landscape that no longer supports T'exelc values and is more likely to burn at uncharacteristic high severities. This nuanced understanding of the Indigenous contribution to a mixed-severity fire regime is critical for advancing proactive fire mitigation that is ecoculturally relevant and guided by Indigenous expertise.

Efficient aqueous molybdenum removal using commercial Douglas fir biochar and its iron oxide hybrids.

Molybdenum (Mo) is a naturally-occurring trace element in drinking water. Most commonly, molybdate anions (MoO42-) are in well water and breast milk. In addition, it is used in medical image testing. Recently, the EPA classified Mo as a potential contaminant, as exposure can lead to health effects such as gout, hyperuricemia, and even lung cancer. We have assessed the sorptive removal of aqueous molybdate using Douglas fir biochar (DFBC) and a hybrid DFBC/Fe3O4 composite containing chemically-coprecipitated iron oxide (Fe3O4). Adsorption was studied at various: pH values, equilibrium times (5 min-24 h), initial Mo concentrations (2.5-1000 mg/L), and temperatures (5, 25, and 40 °C) using batch sorption and fixed-bed column equilibrium methods. Langmuir capacities for DFBC and DFBC/Fe3O4 (at pH 3, 2 hrs equilibrium) were within 459.3-487.9 mg/g and 288-572 mg/g, respectively. These adsorbents and their Mo-laden counterparts were characterized by elemental analysis, BET, PZC, SEM, TEM, EDS, XRD, and XPS. MoO42- adsorption on DFBC is thought to be governed primarily via electrostatic attraction. Adsorption by DFBC/Fe3O4 is primarily governed by chemisorption onto magnetite surface hydroxyl groups, while electrostatics prevail in the DFBC-exposed phase. Stoichiometric precipitation of iron molybdates triggered by iron dissolution was also considered. The data suggest that DFBC and DFBC/Fe3O4 are promising candidates for molybdate sorption.

Comparative Antioxidant and Antimicrobial Activities of Several Conifer Needles and Bark Extracts.

Nowadays, an increased concern regarding using natural products for their health benefits can be observed. The aim of this study was to assess and compare several phenolic compounds found in 15- to 60-year-old Douglas fir, silver fir, larch, pine, and spruce needle and bark extracts and to evaluate their antioxidant and antimicrobial activities. Spectrophotometric assays were used to determine the total polyphenol content and the antioxidant activity that was assessed by using the DPPH• radical scavenging assay (RSA), the ferric reducing antioxidant power assay (FRAP), and the ABTS•+ radical cation scavenging assay (ABTS). The phytochemical content was determined by using high-performance liquid chromatography, and the antimicrobial activity was determined by assessing the minimal inhibition concentration (MIC). The results of the study show a total polyphenol content of 62.45-109.80 mg GAE/g d.w. and an antioxidant activity of 91.18-99.32% for RSA, 29.16-35.74 µmol TE/g d.w. for FRAP, and 38.23-53.57 µmol TE/g d.w. for ABTS. The greatest quantity of phenolic compound for most of the extracts was for (+)-catechin, and it had values between 165.79 and 5343.27 µg/g d.w. for these samples. The antimicrobial inhibition for all the extracts was the strongest for Staphylococcus aureus (MIC 62.5-125 µg/mL). The extracts analyzed could be used for their bioactive potential after further investigations.

Evaluation of the Shear Performance of Douglas-Fir Wood at Elevated Temperatures.

Shear fracture frequently occurs in timber beams and panels subjected to transverse loads. At elevated temperatures, wood will undergo complex physical and chemical processes which significantly affect the shear properties. In this paper, the v-notched Douglas-fir specimens with three different shear planes: (a) Radial-Tangential (RT); (b) Radial-Longitudinal (RL), and (c) Longitudinal-Radial (LR), were fabricated and tested under the elevated temperatures from 20 °C to 180 °C. The digital image correlation (DIC) technique was used to measure the shear strain. It was found that the shear plane had a significant effect on the failure modes, shear strength, and shear modulus. The shear strength and shear modulus generally decreased with the increase of temperature. However, the shear strength was significantly improved when the hardening of the dry lignin occurred between 100 °C and 140 °C. Moreover, the design curve for the shear strength in Eurocode 5 is conservative for all the specimens with different shear planes.

High consistency of trophic niches in generalist arthropod species (Oribatida, Acari) across soil depth and forest type.

Many traits including trophic niche parameters are attributed to species. However, generalist species may vary in trophic niches with environments, making species-based knowledge hard to extrapolate beyond local food webs. Here we tested trophic consistency in oribatid mite species (Acari), one of the most abundant arthropods that occupy all trophic levels in soil food webs. We used stable isotope analysis to compare trophic niches of 40 Oribatida species that co-occur in litter (OL) and soil (0-5 cm, mainly OF/H, AH) of five forest types (native European beech, non-native Douglas fir, range-expanding Norway spruce, two beech-conifer mixed forests). Although stable isotope signatures of bulk material differed between litter and soil, 13C and 15 N values of Oribatida species were remarkably stable irrespective of soil depth. Furthermore, Oribatida were more enriched in 13C in European beech than in coniferous forests, but forest type little affected 15 N values of Oribatida across a range of site conditions. We conclude that Oribatida species occupy virtually identical trophic niches (δ13C and δ15N values) irrespective of the soil depth they colonize and that forest management including non-native tree species little affects trophic position (δ15N values) of oribatid mites. Our findings suggest that the trophic position can be used as a trait in community analysis of Oribatida across forest ecosystems. Our results further indicate that trophic niches of generalist species can be highly consistent irrespective of environment.

Distribution of a Foliage Disease Fungus Within Canopies of Mature Douglas-Fir in Western Oregon.

Nothophaeocryptopus gaeumannii is a common native, endophytic fungus of Douglas-fir foliage, which causes Swiss needle cast, an important foliage disease that is considered a threat to Douglas-fir plantations in Oregon. Disease expression is influenced by fungal fruiting bodies (pseudothecia), which plug the stomata and inhibit gas exchange. Trees are impacted when pseudothecia plug stomates on 1-year-old and older needles resulting in early needle abscission. Mature (100 years+) trees appear to be less impacted from disease, and we hypothesize this is due to the greater emergence of pseudothecia on older than younger needles, which allows for more needle retention. We measured the density of pseudothecia occluding stomates across 2- to 5-year-old needles from upper, middle, and lower canopy positions of mature trees at three sites in the Oregon Coast Range and two sites in the western Oregon Cascade Mountains. Binomial generalized linear mixed model (GLMM) was used to test for the effects of canopy position (upper, middle, and lower), sites, needle age (2-5 years old), and years (2016 and 2017), and their interactions on the pseudothecia density. Pseudothecia density varied annually depending on sites, needle age and canopy positions. Pseudothecia density peaked on 3-, and 4-year-old needles, however, needles emerging from the same year, like 2-year-old needles in 2016 and 3-year-old needles in 2017 both emerged in 2014, had consistently similar patterns of pseudothecia density for both years, across site and canopy positions. Canopy position was important for 3-, and 4-year-old needles, showing less pseudothecia in the lower canopy. This research confirms that N. gaeumannii pseudothecia density is greatest in 3- and 4-year old needles in mature trees in contrast to plantations where pseudothecia density usually peaks on 2-year-old needles, and that pseudothecia density (disease severity) is generally lower in mature trees. Something about mature forest canopies and foliage appears to increase the time it takes for pseudothecia to emerge from the needles, in contrast to younger plantations, thus allowing the mature trees to have greater needle retention.