Linda E. Tackaberry

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.

Mycorrhizal ecology on serpentine soils

Background: Serpentine ecosystems support different, often unique, plant communities; however, we know little about the soil organisms that associate with these ecosystems. Mycorrhizas, mutualistic symbioses between fungi and roots, are critical to nutrient cycling and energy exchange below ground. Aims: We address three hypotheses: H1, diversity of mycorrhizal fungi in serpentine soils mirrors above-ground plant diversity; H2, the morphology of mycorrhizas and fungi on serpentine soils differs from that on non-serpentine; and H3, mycorrhizal fungal communities of the same or closely related hosts differ between serpentine and non-serpentine soils. Methods: This review focuses on whether plant diversity on serpentine soils correlates with the below ground diversity of mycorrhizal fungi. Results: Studies show that plants and fungi formed abundant ectomycorrhizal and arbuscular mycorrhizal symbioses on and off serpentine soils. No serpentine-endemic fungi were identified. Molecular analyses indicate distinct serpentine isolates for Cenococcum geophilum and for Acaulospora, suggesting adaptation to serpentine soils. While fungal sporocarp assemblages on serpentine sites resembled those off serpentine, fruiting of hypogeous fungi was greatly reduced. Conclusions: Ectomycorrhizal fungal communities did not differ between soil types; however, arbuscular mycorrhizal communities differed in some cases but not others. The additive response to multiple factors, described as the serpentine syndrome, may explain part of the response by fungi.

Ectomycorrhizae establishment on Douglas-fir seedlings following chloropicrin treatment to control laminated-root rot disease: Assessment 4 and 5 years after outplanting

Abstract Laminated-root rot, caused by Phellinus weirii (Murr.) Gilb., is a serious disease affecting Douglas-fir and other commercially important species of conifers in northwestern North America. Recent work has shown that this fungus is successfully reduced or eliminated by the fumigant chloropicrin. However, the effect of this biocide on nontarget organisms, including ectomycorrhizae, is uncertain. Following an initial assessment of organisms 2 years after application of the fumigant, a reassessment of the establishment of ectomycorrhizae on Douglas-fir seedlings was undertaken 4.5 and 5.5 years following chloropicrin application. Our findings show that in areas around stumps treated with 20% and 100% of the labeled dosage and in areas around non-treated stumps, chloropicrin did not adversely affect the formation of ectomycorrhizae on young Douglas-fir seedlings by naturally occurring fungi. No significant effect on the abundance or type of mycorrhizae were detected. On this site, chloropicrin did not affect these mycorrhizal associations, at least for 5 years following application.

Site properties have a stronger influence than fire severity on ectomycorrhizal fungi and associated N-cycling bacteria in regenerating post-beetle-killed lodgepole pine forests

Following a pine beetle epidemic in British Columbia, Canada, we investigated the effect of fire severity on rhizosphere soil chemistry and ectomycorrhizal fungi (ECM) and associated denitrifying and nitrogen (N)-fixing bacteria in the root systems of regenerating lodgepole pine seedlings at two site types (wet and dry) and three fire severities (low, moderate, and high). The site type was found to have a much larger impact on all measurements than fire severity. Wet and dry sites differed significantly for almost all soil properties measured, with higher values identified from wet types, except for pH and percent sand that were greater on dry sites. Fire severity caused few changes in soil chemical status. Generally, bacterial communities differed little, whereas ECM morphotype analysis revealed ectomycorrhizal diversity was lower on dry sites, with a corresponding division in community structure between wet and dry sites. Molecular profiling of the fungal ITS region confirmed these results, with a clear difference in community structure seen between wet and dry sites. The ability of ECM fungi to colonize seedlings growing in both wet and dry soils may positively contribute to subsequent regeneration. We conclude that despite consecutive landscape disturbances (mountain pine beetle infestation followed by wildfire), the “signature” of moisture on chemistry and ECM community structure remained pronounced.

Structural characteristics of root–fungus associations in two mycoheterotrophic species, Allotropa virgata and Pleuricospora fimbriolata (Monotropoideae), from southwest Oregon, USA

All members of the Monotropoideae (Ericaceae), including the species, Allotropa virgata and Pleuricospora fimbriolata, are mycoheterotrophs dependent on associated symbiotic fungi and autotrophic plants for their carbon needs. Although the fungal symbionts have been identified for A. virgata and P. fimbriolata, structural details of the fungal–root interactions are lacking. The objective of this study was, therefore, to determine the structural features of these plant root–fungus associations. Root systems of these two species did not develop dense clusters of mycorrhizal roots typical of some monotropoid species, but rather, the underground system was composed of elongated rhizomes with first- and second-order mycorrhizal adventitious roots. Both species developed mantle features typical of monotropoid mycorrhizas, although for A. virgata, mantle development was intermittent along the length of each root. Hartig net hyphae were restricted to the host epidermal cell layer, and fungal pegs formed either along the tangential walls (P. fimbriolata) or radial walls (A. virgata) of epidermal cells. Plant-derived wall ingrowths were associated with each fungal peg, and these resembled transfer cells found in other systems. Although the diffuse nature of the roots of these two plants differs from some members in the Monotropoideae, the structural features place them along with other members of the Monotropoideae in the “monotropoid” category of mycorrhizas.

Microbial community structure of soils under four productivity classes of aspen forests in northern British Columbia

Abstract Trembling aspen (Populus tremuloides) is widely distributed in North American forests. Increased stand productivity with resource availability has been reported, but the relationship between soil microbial community structure and stand productivity remains unclear. To examine soil microbial composition of 4 aspen stand productivity classes, we assessed soil properties, microbial biomass and respiration, and bacterial and ectomycorrhizal diversity. Most variables showed no significant differences between productivity classes. However, mean values for basal respiration (0.05 to 27.99 µg CO2-C·g-1 soil·h-1), bacterial biomass, and metabolic quotient (0.08 to 5.22 CO2-C·mg-1 Cmic·h-1) were lowest in low productivity (Class 1) sites. Bacteria to fungi ratios were significantly lower (P = 0.05) in Class 1 compared to other classes. Microbial biomass ranged from 1.39 to 8.11 mg Cmic·g-1 soil. Thirty-seven distinct aspen ectomycorrhizas (ECM) were characterized, 21 were considered rare (from ≤3 trees). ECM richness did not differ significantly between classes, although relative abundance for some types did. Canonical correspondence analysis showed productivity class explained most microbial community variation, e.g., ECM fungi (80% explained) and soil bacteria (46%). Despite some differences, we could not identify statistically significant bacterial or ECM assemblages linked to stand productivity. Results may reflect a strong association between microbial processes and the dominant host, aspen. Aspen associated with widely distributed fungi common to all classes, possibly facilitating its survival and growth, including on sites exhibiting low pH and low soil fertility.

Re-publication of a translation of ‘The vegetative organs of Monotropa hypopitys L.’ published by F. Kamienski in 1882, with an update on Monotropa mycorrhizas

This paper begins with a brief comparison of Franz Kamienski’s 1882 view of the fungus-root associations and nutrition of Monotropa hypopitys with our current understanding. The rest of this paper is a re-publication of Shannon Berch’s 1985 translation of Kamienski’s breakthrough paper in which it was asserted that Monotropa forms a mutualistic symbiosis and is nourished by fungi associated with the roots of neighbouring trees.

Anti-proliferative activity of a purified polysaccharide isolated from the basidiomycete fungus Paxillus involutus

A growth-inhibitory polysaccharide (GIPinv) was purified using size-exclusion and ion-exchange chromatography from the fourth sodium hydroxide extraction step of a fungus found in British Columbia. The fungus was genetically identified as a member of the Paxillus involutus complex. GIPinv has an average molecular weight of 229kDa and is a heteroglycan composed of glucose (65.9%), galactose (20.8%), mannose (7.8%), fucose (3.2%) and xylose (2.3%). GC-MS methylation analysis suggests that GIPinv has mixed linkages in the backbone containing (1→6)-Gal (25.5%), (1→4)-Glc (18.3%), (1→6)-Glc (8.3%), (1→3)-Glc (5.3%) and (1→2)-Xyl (4.5%). GIPinv has branching points at (1→2, 6)-Man (8.6%) and (1→3, 6)-Man (4.9%) having unsubstituted fucose (8.3%) and glucose (16.3%) as terminal sugars. GIPinv had growth-inhibitory activity against several cancer cell lines and triggered apoptosis. GIPinv should be further explored as a potential anti-cancer agent and a unique polysaccharide.

Growth-Inhibitory and Immunomodulatory Activities of Wild Mushrooms from North-Central British Columbia (Canada)

Wild mushrooms, especially from North America, have not been systematically explored for their medicinal properties. Here we report screening for the growth-inhibitory and immunomodulatory activities of 12 species collected from multiple locations in north-central British Columbia, Canada. Mushrooms were characterized using morphology and DNA sequencing, followed by chemical extraction into 4 fractions using 80% ethanol, 50% methanol, water, and 5% sodium hydroxide. Growth-inhibitory, immunostimulatory, and anti-inflammatory activities of 5 mushrooms (Leucocybe connata, Trichaptum abietinum, Hydnellum sp., Gyromitra esculenta, and Hericium coralloides) are reported here, to our knowledge for the first time. Growth-inhibitory effects were assessed using the cytotoxic MTT assay. Immunostimulatory activity was assessed by tumor necrosis factor-α production in Raw 264.7 macrophages, whereas anti-inflammatory activity was assessed based on the inhibition of lipopolysaccharide-induced tumor necrosis factor-α production. The ethanol and aqueous extracts of Hydnellum sp. were potent growth inhibitors, with a half-maximal inhibitory concentration of 0.6 mg/mL. All 5 fungi displayed strong immunostimulatory activity, whereas only L. connata and T. abietinum showed strong anti-inflammatory activity. For the 7 other fungi investigated, which included well-known medicinal species such as Inonotus obliquus, Phellinus igniarius, and Ganoderma applanatum, the remarkable similarities in the biological activities reported here, and by others for specimens collected elsewhere, suggest that mushrooms can produce similar metabolites regardless of their habitat or ecosystem. This is to our knowledge the first study to explore wild mushrooms from British Columbia for biological activities that are relevant to cancer, and the results provide an initial framework for the selection of mushroom species with the potential for discovery of novel anticancer compounds.

Lichens Contribute to Open Woodland Stability in the Boreal Forest Through Detrimental Effects on Pine Growth and Root Ectomycorrhizal Development

In the boreal forest, open lichen woodlands have been described as an alternative stable state to closed-crown feather moss forest. In this study, we addressed the role of terricolous lichens in stabilizing open woodlands by hindering tree regeneration and/or growth. Based on field and greenhouse experiments, we compared germination and growth of jack pine (Pinus banksiana) on feather mosses (primarily Pleurozium schreberi) and lichens (primarily Cladonia stellaris), using bare mineral soil as a control. Drivers were investigated by (1) manipulating nutrient supply, (2) simulating shade of a closed canopy on the ground layer with the assumption this would mitigate lichen influence on pine growth, and (3) examining pine root ectomycorrhizal colonization and diversity as indicators of pine ability to take up nutrients. Total growth of 6-month-old greenhouse and 2–3-year-old field seedlings, as well as belowground growth of 2-year-old greenhouse seedlings, was significantly greater in moss than in lichen. Seed germination was not affected by ground cover type. Although field phosphorus and base cation availability was greater in mosses than in lichens, fertilization did not entirely compensate for the negative effects of lichens on pine growth in the greenhouse. Ground layer shading had no impact on pine growth. Lichens were associated with reduced abundance and modified composition of the root ectomycorrhizal community. By suggesting that terricolous lichens constitute a less favorable growth substrate than mosses for pine, our results support the hypothesis that lichens contribute to open woodland stability in the potentially closed-crown feather moss forest.