New species and reports of Cuphophyllus from northern North America compared with related Eurasian species.

This study describes four gray or brown species of Cuphophyllus (Hygrophoraceae, Agaricales), two of them new species, restricted to arctic-alpine and northern boreal zones of North America, and relates them morphologically and phylogenetically using multigene and nuc rDNA internal transcribed spacer ITS1-5.8S-ITS (ITS barcode) analyses to their similar, known counterparts. Cuphophyllus cinerellus, epitypified here, is shown to be a pan-palearctic species with sequence-confirmed collections from Fennoscandia and easternmost Asia. Occupying a similar habitat in the Nearctic is its sister species, the morphologically similar but novel C. esteriae, so far known only from eastern North America, including Greenland. Sister to the C. cinerellus-C. esteriae lineage, and known only from boreal raised Sphagnum bogs in Newfoundland, is a new medium-sized light cinereous brown species, C. lamarum. It has a yellow stipe but is phylogenetically distant from the yellow-stiped European C. flavipes and its North American sister species, Hygrophorus pseudopallidus. As cryptic speciation was discovered within C. flavipes, we lecto- and epitypify the name and transfer H. pseudopallidus to Cuphophyllus based on ITS analysis of the holotype. We also transfer the small European Hygrocybe comosa to Cuphophyllus based on morphology. Cuphophyllus hygrocyboides is reported from North America with the first sequence-confirmed collections from arctic-alpine British Columbia and Greenland. In addition, sequencing the holotype of C. subviolaceus identifies it as the sister species to the putative C. lacmus. Both species seem to have an intercontinental distribution. In total, we add new sequences to GenBank from 37 Cuphophyllus collections, including the holotypes of C. hygrocyboides and C. subviolaceus, the two new epitypes, and the two novel species.

Truffle diversity (Tuber, Tuberaceae) in British Columbia.

To improve baseline data for the developing truffle industry in British Columbia, we compiled existing Tuber species sequences from published and unpublished studies and generated new ITS sequences for truffles belonging to Tuber collected in the province. In doing so, we obtained evidence that 13 species of Tuber occur in the province, including six introduced and seven native species, two of which are putative undescribed species. Of the native species, the Tuber anniae species complex is widely distributed in the province while Tuber beyerlei appears to be much more restricted in distribution. Four of the introduced species have commercial value (Tuber melanosporum, Tuber aestivum, Tuber brumale, and Tuber borchii) as do two of the native species (Tuber gibbosum and Tuber oregonense). Focused sampling on likely tree hosts, both hardwood and Pinaceae species, as well as in currently unexplored parts of the province seems likely to expand our knowledge of the diversity and distribution of Tuber species in British Columbia.

Cultivation of Mediterranean species of Tuber (Tuberaceae) in British Columbia, Canada.

Based on an assessment of soil and climatic conditions in British Columbia (BC), the Truffle Association of British Columbia (TABC) determined that the cultivation of Mediterranean Tuber melanosporum and Tuber aestivum might be possible in the warmer parts of the province. With the cooperation of independent truffle growers, TABC assessed the colonization of host tree roots collected from eight truffle orchards planted 2-7 years earlier using morphological and molecular criteria. Both Tuber species persisted on the roots of inoculated trees in six of the eight truffle orchards studied. The identity of Tuber ectomycorrhizas that had been characterized morphologically as differing from those of T. melanosporum and T. aestivum were determined using DNA sequence analysis to belong to three species of truffles native to the Pacific Northwest. One of those species, Tuber anniae, had been previously reported from BC, but the other two, Tuber menseri nom. prov. and Tuber beyerlei, are reported here from BC for the first time. Recently, production of three Périgord black truffles in one truffle orchard and one Burgundy truffle in another orchard demonstrates that these truffles are able to fruit in BC.

Effects of growth medium, nutrients, water, and aeration on mycorrhization and biomass allocation of greenhouse-grown interior Douglas-fir seedlings.

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.

The effect of fertilization on the below-ground diversity and community composition of ectomycorrhizal fungi associated with western hemlock (Tsuga heterophylla).

Fertilization typically reduces ectomycorrhizal diversity shortly after its application but less is known about its longer-term influence on fungal species. Long-term effects are important in forests where fertilizer is rarely applied. We compared fungal species composition in western hemlock control plots with plots last fertilized 7 years ago with nitrogen (N) or nitrogen plus phosphorus (N + P). The N + P fertilization had a significant lingering effect, increasing the tree size and foliar P content of the western hemlocks. From ectomycorrhizal roots of 24-year-old trees from northern Vancouver Island, Canada, we identified fungi from 12 samples per treatment, by amplifying, cloning, and sequencing fungal ribosomal DNA fragments, placing sequences with 97% or more identity in the same operational taxonomic unit (OTU). Diversity was high across treatments; we detected 77 fungal OTUs, 52 from ectomycorrhizal genera, among 922 clone sequences. The five most frequent OTUs were similar in abundance across treatments. Only 19 OTUs matched any of the 197 previously reported ectomycorrhizal species of western hemlock. Species composition but not diversity in nitrogen plus phosphorus plots differed significantly from control or nitrogen plots. Two Cortinarius OTUs were indicator species for nitrogen plus phosphorus plots and presence of Cortinarius cinnamomeus was correlated with control or nitrogen plots. After 7 years, fertilization history had made no detectable difference in ectomycorrhizal fungal diversity, but long-lasting changes in environment resulting from fertilization had a lingering effect on fungal ectomycorrhizal species composition.

Culturing and direct DNA extraction find different fungi from the same ericoid mycorrhizal roots.

• This study compares DNA and culture-based detection of fungi from 15 ericoid mycorrhizal roots of salal (Gaultheria shallon), from Vancouver Island, BC Canada. • From the 15 roots, we PCR amplified fungal DNAs and analyzed 156 clones that included the internal transcribed spacer two (ITS2). From 150 different subsections of the same roots, we cultured fungi and analyzed their ITS2 DNAs by RFLP patterns or sequencing. We mapped the original position of each root section and recorded fungi detected in each. • Phylogenetically, most cloned DNAs clustered among Sebacina spp. (Sebacinaceae, Basidiomycota). Capronia sp. and Hymenoscyphus erica (Ascomycota) predominated among the cultured fungi and formed intracellular hyphal coils in resynthesis experiments with salal. • We illustrate patterns of fungal diversity at the scale of individual roots and compare cloned and cultured fungi from each root. Indicating a systematic culturing detection bias, Sebacina DNAs predominated in 10 of the 15 roots yet Sebacina spp. never grew from cultures from the same roots or from among the > 200 ericoid mycorrhizal fungi previously cultured from different roots from the same site.