Keith N. Egger

The effect of Kalmia angustifolia on the growth, nutrition, and ectomycorrhizal symbiont community of black spruce

Abstract Kalmia angustifolia L. is an ericaceous shrub that frequently invades black spruce (Picea mariana Mill.) clear-cuts in central Newfoundland. Field observations suggest that on many sites where K. angustifolia grows, black spruce seedlings become chlorotic and stunted. Previous laboratory research has suggested that allelochemicals of K. angustifolia affect the growth and development of black spruce as well as the growth of certain ectomycorrhizal (ECM) fungi associated with black spruce. Black spruce seedlings close to ( 1 m) K. angustifolia were sampled from a clear-cut in central Newfoundland. The ECM community structure, degree of mycorrhizal infection, height, mass, root:shoot ratio, and the foliar concentrations of N, P, K, Ca, and Mg of spruce seedlings growing close to and far from K. angustifolia were examined. Seedlings close to K. angustifolia had significantly lower foliar concentrations of N and P, had a lower rate of mycorrhizal infection, and were more frequently associated with Phialocephala dimorphospora Kendrick, a potential root pathogen of black spruce, than seedlings growing far from K. angustifolia. There were positive linear relationships between black spruce foliar N concentration and total seedling height and biomass for seedlings growing away from K. angustifolia, but not for seedlings in close proximity to K. angustifolia. Hypotheses suggesting possible roles for nutrient competition, allelopathy, and K. angustifolias ability to increase the occurrence of the pseudomycorrhizal P. dimorphospora on black spruce are proposed.

Ectendomycorrhizal associations - characteristics and functions

Abstract. Mycorrhizal symbioses are widespread mutualistic associations of many plant hosts found in many habitats. One type of putative mycorrhizal association, ectendomycorrhiza, is confined to Pinus and Larix spp. and is common in conifer nurseries and in disturbed habitats. This association is characterized by the unique combination of a fungal mantle, Hartig net, and intracellular hyphae, the latter forming soon after Hartig net development. Many reports of the occurrence of ectendomycorrhizas from field-collected specimens are likely erroneous and instead may represent senescent ectomycorrhizas. The fungus species involved in the formation of ectendomycorrhizas were initially called E-strain fungi and their identification was based on characteristics of hyphae and chlamydospores. With the discovery of teleomorphs for some of these fungi, they were found to be ascomycetes. More recently, molecular methods have been used to clarify their systematics and phylogeny and it is apparent that most of the isolates belong to two species, Wilcoxina mikolae and Wilcoxina rehmii. Two species of dematiaceous fungi and a member of the Pezizales, Sphaerosporella brunnea, also have been reported to form ectendomycorrhizas. These fungi can form ectendomycorrhizas with their hosts over a broad pH range and may utilize many substrates as a carbon source. Ectendomycorrhizas may be important in the revegetation of disturbed sites and in the establishment of conifer seedlings in post-fire situations.

The Genus Oidiodendron: Species Delimitation and Phylogenetic Relationships Based on Nuclear Ribosomal DNA Analysis

Nuclear ribosomal DNA sequences (ITS region) of fifteen species in the hyphomycete genus Oidiodendron and ten species from the 4 genera in the Myxotrichaceae, Byssoascus, Gymnostellatospora, Myxotrichum, and Pseudogymnoascus, were analysed to: (i) reveal the levels of intra- versus interspecific sequence variation within the genus Oidiodendron, clarify species delimitation and examine the useful- ness of some morphological characters used for iden- tification; (ii) assess the possible conspecificity of doc- umented ericoid mycorrhizal strains of Oidiodendron; and (iii) test the hypothesis based on morphological inference that the genus Oidiodendron belongs with the genus Myxotrichum in the Myxotrichaceae (Ony- genales). Comparison of molecular and morpholog- ical data for multiple strains of 0. griseum, 0. tenuis- simum and 0. maius revealed that conidiophore length and the production of a diffusing pigment are not reliable key characters for the genus. Several his- torically important ericoid mycorrhizal strains, doc- umented as 0. griseum, were reidentified as 0. maius. Parsimony analyses of 23 Oidiodendron strains showed that three highly supported monophyletic groups, each one consisting of a pair of species, are resolved within the genus. A low level of sequence divergence between the species in these pairs suggests conspe- cificity for each pair. Other interspecific relationships were not well-supported by bootstrap values. Parsi- mony analysis of a second dataset composed of mi- totic and meiotic taxa showed that Oidiodendron, My- xotrichum and Byssoascus form a well-supported monophyletic group within the Myxotrichaceae, and

Long-term experimental warming alters nitrogen-cycling communities but site factors remain the primary drivers of community structure in high arctic tundra soils

Arctic air temperatures are expected to rise significantly over the next century. Experimental warming of arctic tundra has been shown to increase plant productivity and cause community shifts and may also alter microbial community structure. Hence, the objective of this study was to determine whether experimental warming caused shifts in soil microbial communities by measuring changes in the frequency, relative abundance and/or richness of nosZ and nifH genotypes. Five sites at a high arctic coastal lowland were subjected to a 13-year warming experiment using open-top chambers (OTCs). Sites differed by dominant plant community, soil parent material and/or moisture regimen. Six soil cores were collected from each of four replicate OTC and ambient plots at each site and subdivided into upper and lower samples. Differences in frequency and relative abundance of terminal restriction fragments were assessed graphically by two-way cluster analysis and tested statistically with permutational multivariate analysis of variance (ANOVA). Genotypic richness was compared using factorial ANOVA. The genotype frequency, relative abundance and genotype richness of both nosZ and nifH communities differed significantly by site, and by OTC treatment and/or depth at some sites. The site that showed the most pronounced treatment effect was a wet sedge meadow, where community structure and genotype richness of both nosZ and nifH were significantly affected by warming. Although warming was an important factor affecting these communities at some sites at this high arctic lowland, overall, site factors were the main determinants of community structure.

Substrate hydrolysis patterns of post-fire Ascomycetes (Pezizales)

Forty-one isolates of post-fire Ascomycetes, representing 1 species of Sphaeriales and 25 species of Pezizales, were tested for ability to hydrolyze amylose, amylopectin, gelatin, xylan, lipid, pectin, chitin, cellulose, and lignin. Isolates were also assayed for the presence of phenol oxidase enzymes: laccase, tyrosinase and peroxidase. The majority of isolates were able to degrade amylose, amylopectin, gelatin, and xylan, and were unable to utilize chitin. Cellulose, lignin, and pectin utilization showed the least variation between isolates of the same species. Pectin hydrolysis and phenol oxidase production were strongly influenced by the pH of the medium. Phenol oxidase production was identified in both saprotrophs and biotrophs. However, lignin degradation was primarily observed in the saprotrophs. It is hypothesized that phenol oxidases in post-fire Pezizales function in lignin degradation in saprotrophs, but function primarily in detoxification of host-produced phenols in biotrophs. Tests were able to identify guilds of fungi associated with non-lignified substrates (e.g. litter, fine roots) and lignified substrates (e.g. wood, woody roots). Tests also helped distinguish between saprotrophic and biotrophic modes of nutrition. The majority of post-fire Pezizales appear to be opportunistic decomposers or facultative biotrophs adapted to the transient post-fire environment.

The effect of experimental warming on the root-associated fungal community of Salix arctica.

The effect of experimental warming on the root-associated fungal community of arctic willow (Salix arctica) was studied in three distinct habitats at a tundra site in the Canadian High Arctic. Plots were passively warmed for 5–7 years using open-top chambers and compared to control plots at ambient temperature. Fungal communities were assessed using terminal restriction fragment length polymorphisms. We found the following: (1) the root-associated fungal community in these high arctic tundra habitats is highly diverse; (2) site and soil characteristics are the most important drivers of community structure and (3) warming increased the density of different genotypes on individual root sections but has not (yet) affected the composition, richness or evenness of the community. The change in genotype density in the warmed plots was associated with an increase in PCR amplification efficiency, suggesting that increased C allocation belowground is increasing the overall biomass of the fungal community.

Molecular diversity of nifH genes from bacteria associated with high arctic dwarf shrubs.

Biological nitrogen fixation is the primary source of new N in terrestrial arctic ecosystems and is fundamental to the long-term productivity of arctic plant communities. Still, relatively little is known about the nitrogen-fixing microbes that inhabit the soils of many dominant vegetation types. Our objective was to determine which diazotrophs are associated with three common, woody, perennial plants in an arctic glacial lowland. Dryas integrifolia, Salix arctica, and Cassiope tetragona plants in soil were collected at Alexandra Fiord, Ellesmere Island, Canada. DNA was extracted from soil and root samples and a 383-bp fragment of the nifH gene amplified by the polymerase chain reaction. Cloned genotypes were screened for similarity by restriction fragment length polymorphism (RFLP) analysis. Nine primary RFLP phylotypes were identified and 42 representative genotypes selected for sequencing. Majority of sequences (33) were type I nitrogenases, whereas the remaining sequences belonged to the divergent, homologous, type IV group. Within the type I nitrogenases, nifH genes from posited members of the Firmicutes were most abundant, and occurred in root and soil samples from all three plant species. nifH genes from posited Pseudomonads were found to be more closely associated with C. tetragona, whereas nifH genes from putative alpha-Proteobacteria were more commonly associated with D. integrifolia and S. arctica. In addition, 12 clones likely representing a unique clade within the type I nitrogenases were identified. To our knowledge, this study is the first to report on the nifH diversity of arctic plant-associated soil microbes.

Taxonomy and population structure of E-strain mycorrhizal fungi inferred from ribosomal and mitochondrial DNA polymorphisms

E-strain fungi form ecto- and ectendomycorrhizal associations with many trees, and are important mycorrhizal symbionts in disturbed forest habitats and conifer nurseries. Unlike the majority of mycorrhizal fungi, which are basidiomycetes, E-strain fungi are ascomycetes belonging to the genus Wilcoxina (Pezizales). We analysed variation in the nuclear and mitochondrial ribosomal RNA genes to elucidate species concepts among E-strain fungi and to examine their population structure. We found that most E-strain isolates can be assigned to two taxa, W. mikolae and W. rehmii, that have different habitat preferences. Wilcoxina mikolae is the predominant taxon in disturbed forest habitats on soils that are often low in organic matter, such as burned sites, while W. rehmii prefers peaty soils. Analysis of mitochondrial DNA also revealed that within each species, isolates could be differentiated based upon host preference. This is the first report of population subdivision based upon host in E-strain fungi.

Monotropa uniflora: morphological and molecular assessment of mycorrhizae retrieved from sites in the Sub-Boreal Spruce biogeoclimatic zone in central British Columbia

Plant species in the subfamily Monotropoideae are achlorophyllous and have developed a complex mode of nutrition, receiving photosynthates from neighboring trees via shared fungi. To explore the mycorrhizal associations of Monotropa uniflora in central British Columbia (B.C.), plants were sampled from three sites: a Betula-dominated site and two sites with a mixture of conifer and hardwood trees. Fifteen M. uniflora root-clusters were sampled (five per site) and the mycorrhizal diversity was assessed using morphological and molecular (PCR-RFLP analysis and DNA sequencing) methods. Both methods showed that root-clusters (often comprising several hundred mycorrhizal tips) belonging to the same plant appeared to involve fungus monocultures in the family Russulaceae. All mycorrhizae exhibited typical Russula morphology and had mantle cystidia. Two root-clusters, one each from sites 1 and 3, lacked one of the two types of cystidia present on all other root-clusters. PCR-RFLP analysis resulted in three fragment patterns for the 15 root clusters. One molecular fragment pattern included the two root-clusters displaying the single cystidium type plus an additional root-cluster with both cystidia types. DNA sequencing of a portion of the ITS2 region of the ribosomal DNA suggests that the three variants represent different species; two of the variants clustered with the hypogeous fungi Martellia and Gymnomyces. The study provides increased evidence of low diversity and high specificity in the Monotropa-fungus relationship and suggests that M. uniflora associates uniquely with fungi in the family Russulaceae in central B.C.

Host and habitat filtering in seedling root-associated fungal communities: taxonomic and functional diversity are altered in ‘novel’ soils

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.