Daniel M. Durall

Net transfer of carbon between ectomycorrhizal tree species in the field

Different plant species can be compatible with the same species of mycorrhizal fungi, and be connected to one another by a common mycelium,. Transfer of carbon, nitrogen, and phosphorus, through interconnecting mycelia has been measured frequently in laboratory experiments, but it is not known whether transfer is bidirectional, whether there is a net gain by one plant over its connected partner, or whether transfer affects plant performance in the field,. Laboratory studies using isotope tracers show that the magnitude of one-way transfer can be influenced by shading of ‘receiver’ plants,, fertilization of ‘donor’ plants with phosphorus, or use of nitrogen-fixing donor plants and non-nitrogen-fixing receiver plants,, indicating that movement may be governed by source–sink relationships. Here we use reciprocal isotope labelling in the field to demonstrate bidirectional carbon transfer between the ectomycorrhizal tree species Betula papyrifera and Pseudotsuga menziesii, resulting in net carbon gain by P. menziesii. Thuja plicata seedlings lacking ectomycorrhizae absorb small amounts of isotope, suggesting that carbon transfer between B. papyrifera and P. menziesii is primarily through the direct hyphal pathway. Net gain by P. menziesii seedlings represents on average 6% of carbon isotope uptake through photosynthesis. The magnitude of net transfer is influenced by shading of P. menziesii, indicating that source–sink relationships regulate such carbon transfer under field conditions.

Carbon and Nutrient Fluxes Within and Between Mycorrhizal Plants

Mycorrhizal fungi are involved in the uptake of nutrients in exchange for C from host plants, and possibly in the transfer of C and nutrients between plants. Ecto-mycorrhizal fungi (EMF) increase uptake rates of nutrients by a variety of mechanisms, including increased physical access to soil, changes to mycorrhizosphere or hyphosphere chemistry, and alteration of the bacterial community in the mycorrhizosphere. They influence mycorrhizosphere chemistry through release of organic acids and production of enzymes. Movement of nutrients within an ecto-mycorrhizal (EM) mycelial network, as well as exchange of C and nutrients between symbionts, appear to be regulated by source-sink relationships. Estimates of the quantity of plant C partitioned belowground (to roots and EMF) varies widely (40–73%) depending on the methodology used and ecosystem studied, and is affected by several factors such as the identity of plant and fungal species, plant nutrient content, and EM age.

Architecture of the wood-wide web: Rhizopogon spp. genets link multiple Douglas-fir cohorts.

*The role of mycorrhizal networks in forest dynamics is poorly understood because of the elusiveness of their spatial structure. We mapped the belowground distribution of the fungi Rhizopogon vesiculosus and Rhizopogon vinicolor and interior Douglas-fir trees (Pseudotsuga menziesii var. glauca) to determine the architecture of a mycorrhizal network in a multi-aged old-growth forest. *Rhizopogon spp. mycorrhizas were collected within a 30 x 30 m plot. Trees and fungal genets were identified using multi-locus microsatellite DNA analysis. Tree genotypes from mycorrhizas were matched to reference trees aboveground. Two trees were considered linked if they shared the same fungal genet(s). *The two Rhizopogon species each formed 13-14 genets, each colonizing up to 19 trees in the plot. Rhizopogon vesiculosus genets were larger, occurred at greater depths, and linked more trees than genets of R. vinicolor. Multiple tree cohorts were linked, with young saplings established within the mycorrhizal network of Douglas-fir veterans. A strong positive relationship was found between tree size and connectivity, resulting in a scale-free network architecture with small-world properties. *This mycorrhizal network architecture suggests an efficient and robust network, where large trees play a foundational role in facilitating conspecific regeneration and stabilizing the ecosystem.

Diversity and species distribution of ectomycorrhizal fungi along productivity gradients of a southern boreal forest.

Coniferous forests with diverse ectomycorrhizal fungus (EMF) communities are associated with nutrient-poor, acidic soils but there is some debate whether EMF can be equally adapted to more productive, nitrogen-rich sites. We compared EMF species distribution and diversity along a replicated productivity gradient in a southern boreal forest of British Columbia (Canada). Roots from subalpine fir (Abies lasiocarpa) saplings of the understory were sampled and EMF species were identified by morphotypes supplemented with ITS rDNA analysis. There were significant changes in the distribution and abundance of 74 EMF species along the productivity gradient, with as little as 24% community similarity among contrasting sites. Species richness per plot increased asymptotically with foliar nitrogen concentrations of subalpine fir, demonstrating that many EMF species were well suited to soils with high rates of nitrogen mineralization. EMF species abundance in relation to site productivity included parabolic, negative linear, and positive exponential curves. Both multi-site and more narrowly distributed EMF were documented, and a diverse mix of mantle exploration types was present across the entire productivity gradient. The results demonstrate strong associations of EMF fungal species with edaphic characteristics, especially nitrogen availability, and a specialization in EMF communities that may contribute to the successful exploitation of such contrasting extremes in soil fertility by a single tree host.

Carbon allocation and carbon transfer between t Betula papyrifera and t Pseudotsuga menziesii seedlings using a 13C pulse-labeling method

Here we describe a simple method for pulse-labeling tree seedlings with 13CO2(gas), and then apply the method in two related experiments: t (i) comparison of carbon allocation patterns between t Betula papyrifera Marsh. and t Pseudotsuga menziesii (Mirb.) Franco, and t (ii) measurement of one-way belowground carbon transfer from t B. papyrifera to t P. menziesii. Intraspecific carbon allocation patterns and interspecific carbon transfer both influence resource allocation, and consequently development, in mixed communities of t B. papyrifera and t P. menziesii.In preparation for the two experiments, we first identified the appropriate 13CO2(gas) pulse-chase regime for labeling seedlings: a range of pulse (100-mL and 200-mL 99 atom%13 CO2(gas)) and chase (0, 3 and 6 d) treatments were applied to one year-old t B. papyrifera and t P. menziesii seedlings. The amount of 13CO2 fixed immediately after 1.5 h exposure was greatest for both t B. papyrifera (40.8 mg excess 13C) and t P. menziesii (22.9 mg excess 13C) with the 200-mL pulse, but higher 13C loss and high sample variability resulted in little difference in excess13 C content between pulse treatments after 3 d for either species. The average excess 13C root/shoot ratio of t B. papyrifera and t P. menziesii changed from 0.00 immediately following the pulse to 0.61 and 0.87 three and six days later, which reflected translocation of 75% of fixed isotope out of foliage within 3 d following the pulse and continued enrichment in fine roots over 6 d. Based on these results, the 100-mL CO2(gas) and 6-d chase were considered appropriate for the carbon allocation and belowground transfer experiments.In the carbon allocation experiment, we found after 6 d that t B. papyrifera allocated 49% (average 9.5 mg) and t P. menziesii 41% (average 5.8 mg) of fixed isotope to roots, of which over 55% occurred in fine roots in both species. Species differences in isotope allocation patterns paralleled differences in tissue biomass distribution. The greater pulse labeling efficiency of t B. papyrifera compared to t P. menziesii was associated with its two-fold and 13- fold greater leaf and whole seedling net photosynthetic rates, respectively, 53% greater biomass, and 35% greater root/shoot ratio.For the carbon transfer experiment, t B. papyrifera and t P. menziesii were grown together in laboratory rootboxes, with their roots intimately mingled. A pulse of 100 mL13 CO2(gas) was applied to paper birch and one-way transfer to neighboring t P. menziesii was measured after 6 d. Of the excess 13C fixed by t B. papyrifera, 4.7% was transferred to neighboring t P. menziesii, which distributed the isotope evenly between roots and shoots. Of the isotope received by t P. menziesii, we estimated that 93% was taken up through belowground pathways, and the remaining 7% taken up by foliage as13 CO2(gas) respired by t B. papyrifera shoots. These two experiments indicate that t B. papyrifera fixes more total carbon and allocates a greater proportion to its root system than does t P. menziesii, giving it a competitive edge in resource gathering; however, below-ground carbon sharing is of sufficient magnitude that it may help ensure co-existence of the two species in mixed communities.

Influence of soil nutrients on ectomycorrhizal communities in a chronosequence of mixed temperate forests

Many factors associated with forests are collectively responsible for controlling ectomycorrhizal (ECM) fungal community structure, including plant species composition, forest structure, stand age, and soil nutrients. The objective of this study was to examine relationships among ECM fungal community measures, local soil nutrients, and stand age along a chronosequence of mixed forest stands that were similar in vegetation composition and site quality. Six combinations of age class (5-, 26-, 65-, and 100-year-old) and stand initiation type (wildfire and clearcut) were replicated on four sites, each representing critical seral stages of stand development in Interior Cedar-Hemlock (ICH) forests of southern British Columbia. We found significant relationships between ECM fungal diversity and both available and organic P; available P was also positively correlated with the abundance of two ECM taxa (Rhizopogon vinicolor group and Cenoccocum geophilum). By contrast, ECM fungal diversity varied unpredictably with total and mineralizable N or C to N ratio. We also found that soil C, N, available P, and forest floor depth did not exhibit strong patterns across stand ages. Overall, ECM fungal community structure was more strongly influenced by stand age than specific soil nutrients, but better correlations with soil nutrients may occur at broader spatial scales covering a wider range of site qualities.

Vertical partitioning between sister species of Rhizopogon fungi on mesic and xeric sites in an interior Douglas‐fir forest

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.

The use of propidium monoazide in conjunction with qPCR and Illumina sequencing to identify and quantify live yeasts and bacteria.

Culture-independent methods of microbial identification have been developed, which allow for DNA extraction directly from environmental samples without subjecting microbes to growth on nutrient media. These methods often involve next generation DNA sequencing (NGS) for identifying microbes and qPCR for quantifying them. Despite the benefits of extracting all DNA from the sample, results may be compromised by amplifying DNA from dead cells. To address this short-coming, the use of propidium monoazide (PMA) has been used to deactivate DNA in non-viable cells. Nevertheless, its optimization has not been fully explored under a variety of conditions. In this study, we optimized the PMA method for both yeasts and bacteria. Specifically, we explored the effect different PMA concentrations and different cell densities had on DNA amplification (as part of next generation DNA sequencing) from both dead and viable bacterial and yeast cells. We found PMA was effective in eliminating DNA that was associated with dead yeast and bacterial cells for all cell concentrations. Nevertheless, DNA (extracted from viable yeast and bacterial cells) amplified most abundantly when PMA concentration was at 6μM and when yeast densities ranged between 10(6) to 10(7)CFU/mL and bacterial densities were approximately 10(8)CFU/mL.

Development and use of a quantum dot probe to track multiple yeast strains in mixed culture.

Saccharomyces cerevisiae strains vary in their ability to develop and enhance sensory attributes of alcoholic beverages and are often found growing in mixed strain fermentations; however, quantifying individual strains is challenging due to quantification inaccuracies, low marker longevity, and compromised kinetics. We developed a fluorescent probe, consisting of glutathione molecules conjugated to a quantum dot (QD). Two S. cerevisiae strains were incubated with different coloured probes (QD attached to glutathione molecules, QD-GSH), fermented at multiple ratios, and quantified using confocal microscopy. The QD method was compared with a culture method using microsatellite DNA analysis (MS method). Probes were taken up by an ADP1 encoded transporter, transferred from mother cell to daughter cell, detectable in strains throughout fermentation, and were non-toxic. This resulted in a new quantification method that was more accurate and efficient than the MS method.

Rediscovery of the vesicles that characterized Rhizopogon vesiculosus

Molecular distinction between Rhizopogon vinicolor and R. vesiculosus has been made recently, but the diagnostic “yellow-brown (fresh) inflated cells” of R. vesiculosus, originally described by AH Smith, were not observed. These distinctive hyphal cells (vesicles) have not been reported since the type description. In that description they were said to collapse upon drying and described as being difficult to find. Here we report the rediscovery of these vesicles and describe their specific location on sporocarps of R. vesiculosus. We also report an original discovery that coiled, dark-walled hyphae on the sporocarps and in the mycorrhizae of R. vinicolor are of taxonomic value. The coiled hyphae, combined with the presence or absence of the elusive vesicles, allow R. vesiculosus and R. vinicolor to be morphologically distinguished with increased accuracy.