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When wine grapes are exposed to smoke, there is a risk that the resulting wines may possess smoky, ashy, or burnt aromas, a wine flaw known as smoke taint. Smoke taint occurs when the volatile phenols (VPs) largely responsible for the aroma of smoke are transformed in grape into a range of glycosides that are imperceptible by smell. The majority of VP-glycosides described to date are disaccharides possessing a reducing β-d-glucopyranosyl moiety. Here, a two-part experiment was performed to (1) assess the stability of 11 synthesized VP-glycosides towards general acid-catalyzed hydrolysis during aging, and (2) to examine whether yeast strains differed in their capacity to produce free VPs both from these model glycosides as well as from grapes that had been deliberately exposed to smoke. When fortified into both model and real wine matrices at 200 ng/g, all VP-disaccharides were stable over 12 weeks, while (42-50 ng/g) increases in free 4-ethylphenol and p -cresol were detected when these were added to wine as their monoglucosides. Guaiacol and phenol were the most abundantly produced VPs during fermentation, whether originating from natural VP-precursors in smoked-exposed Pinot Noir must, or due to fortification with synthetic VP-glycosides. Significant yeast strain-specific differences in glycolytic activities were observed for phenyl-β-d-glycopyranoside, with two strains (RC212 and BM45) being unable to hydrolyze this model VP, albeit both were active on the guaiacyl analogue. Thus, differences in Saccharomyces cerevisiae β-glucosidase activity appear to be influenced by the VP moiety.

Saccharomyces cerevisiae is the primary yeast species responsible for most fermentations in winemaking. However, other yeasts, including Saccharomyces uvarum, have occasionally been found conducting commercial fermentations around the world. S. uvarum is typically associated with white wine fermentations in cool-climate wine regions, and has been identified as the dominant yeast in fermentations from France, Hungary, northern Italy, and, recently, Canada. However, little is known about how the origin and genetic diversity of the Canadian S. uvarum population relates to strains from other parts of the world. In this study, a highly diverse S. uvarum population was found dominating uninoculated commercial fermentations of Chardonnay grapes sourced from two different vineyards. Most of the strains identified were found to be genetically distinct from S. uvarum strains isolated globally. Of the 106 strains of S. uvarum identified in this study, four played a dominant role in the fermentations, with some strains predominating in the fermentations from one vineyard over the other. Furthermore, two of these dominant strains were previously identified as dominant strains in uninoculated Chardonnay fermentations at the same winery two years earlier, suggesting the presence of a winery-resident population of indigenous S. uvarum. This research provides valuable insight into the diversity and persistence of non-commercial S. uvarum strains in North America, and a stepping stone for future work into the enological potential of an alternative Saccharomyces yeast species., Competing Interests: The authors have declared that no competing interests exist.

Rhizopogon vesiculosus and R. vinicolor are sister fungal species; they form ectomycorrhizas exclusively with Douglas-fir roots, and they are important in forming relatively large mycorrhizal networks, but they may be vulnerable to disturbance caused by logging practices. The main objective was to determine the resilience of mycorrhizal networks 25 years following removal of large hub trees. We predicted that the targeted removal of mature trees would reduce network connectedness compared with a non-harvested neighboring forest. Rhizopogon vesiculosus was nearly absent in the non-harvested plots, whereas both species were prominent in the harvested plots. Initially, network analysis was based only on networks formed by R. vinicolor because they were well represented in both treatments. These analyses showed that the R. vinicolor-Douglas-fir MN was more densely linked in the non-harvested plots than the harvested plots. When we accounted for differences in link and node density, there was still an edge difference and a greater vulnerability to fragmentation in harvested forests than in non-harvested forests. When both Rhizopogon sister species were included in the analysis, both treatments had similar connectivity and limited vulnerability to fragmentation. This suggests that when these forests transition from a regenerating to a non-regenerating state, the Rhizopogon network will lose R. vesiculosus but will maintain link density due to the colonization with R. vinicolor.

The microbial consortium of wine fermentations is highly dependent upon winemaking decisions made at crush, including the decision to inoculate and the decision to add sulfur dioxide (SO2) to the must. To investigate this, Chardonnay grape juice was subjected to two inoculation treatments (uninoculated and pied de cuve inoculation) as well as two SO2 addition concentrations (0 and 40 mg/L). The bacterial communities, fungal communities and Saccharomyces populations were monitored throughout fermentation using culture-dependent and culture-independent techniques. After fermentation, the wines were evaluated by a panel of experts. When no SO2 was added, the wines underwent alcoholic fermentation and malolactic fermentation simultaneously. Tatumella bacteria were present in significant numbers, but only in the fermentations to which no SO2 was added, and were likely responsible for the malolactic fermentation observed in these treatments. All fermentations were dominated by a genetically diverse indigenous population of Saccharomyces uvarum, the highest diversity of S. uvarum strains to be identified to date; 150 unique strains were identified, with differences in strain composition as a result of SO2 addition. This is the first report of indigenous S. uvarum strains dominating and completing fermentations at a commercial winery in North America., (© FEMS 2019.)

Modern day winemaking often involves the addition of sulfur dioxide (SO 2 ) at crush to act as both an antioxidant and an antimicrobial agent. While the effects of SO 2 on microbial communities and particularly on spoilage microorganisms has been well-studied, the advent of culture-independent molecular technologies, such as Illumina sequencing, allows the subject to be re-visited in a new context. High-throughput amplicon sequencing allows for a more thorough evaluation of microbial communities, as thousands of microbial sequences per sample can be identified and even rare microorganisms can be studied. This research investigated whether the addition of different levels of SO 2 at crush (0, 20, or 40 mg/L) would affect the composition of fungal and bacterial communities, as well as the sensory attributes of the resulting wines. Samples were taken from uninoculated fermentations of Pinot gris and analyzed via high-throughput amplicon sequencing using the Illumina MiSeq platform. Yeast relative abundance and overall fungal community composition differed among the SO 2 additions. Notably, a Hanseniaspora yeast appeared in all treatments and persisted until the end of alcoholic fermentation, although its relative abundance was significantly higher in the fermentations to which low or no SO 2 had been added. Two key wine sensory attributes (citrus aroma and pome fruit flavor) differed among the SO 2 treatments. This research provides an in-depth look into the fungal and bacterial communities during alcoholic fermentation and gives a better understanding of the microbial community response to SO 2 additions during the crush period., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Typically, Mycena species are viewed as saprotrophic fungi. However, numerous detections of Mycena spp. in the roots of green plants suggest that a continuum from saprotrophy to biotrophy could exist. In particular, mycenoid species have repeatedly been found in Ericaceae plant roots. Our study asked whether (1) Mycena species are commonly found in the roots of green Ericaceae plants; (2) Mycena sequences are limited to a single group/lineage within the genus; and (3) a Mycena sp. can behave as a beneficial root associate with a typical ericoid mycorrhizal plant (Vaccinium corymbosum), regardless of how much external labile carbon is available. We detected Mycena sequences in roots of all sampled Ericaceae plants. Our Mycena sequences clustered in four different groups distributed across the Mycena genus. Only one group could be assigned with confidence to a named species (M. galopus). Our Mycena sequences clustered with other Mycena sequences detected in roots of ericoid mycorrhizal plant species collected throughout Europe, America, and Australia. An isolate of M. galopus promoted growth of V. corymbosum seedlings in vitro regardless of external carbon supply in the media. Seedlings inoculated with M. galopus grew as well as those inoculated with the ericoid mycorrhizal fungus Rhizoscyphus ericae. Surprisingly, this M. galopus isolate colonized Vaccinium roots and formed distinctive peg-like structures. Our results suggest that Mycena species might operate along a saprotroph-symbiotic continuum with a range of ericoid mycorrhizal plant species. We discuss our results in terms of fungal partner recruitment by Ericaceae plants.

During winemaking, sulfur dioxide (SO 2 ) is often added prior to the onset of alcoholic fermentation to prevent the growth of spoilage microorganisms and to create an environment that promotes the rapid colonization of the grape must by Saccharomyces cerevisiae. Most recent research has focused on the impacts of SO 2 additions on spoilage microorganisms or on the yeast community at a species level, but less is known about the impacts that SO 2 additions have on S. cerevisiae populations. We investigated whether different levels of SO 2 addition at crush (0, 20, or 40mg/L SO 2 ) have an effect upon the relative abundance and composition of S. cerevisiae strains conducting spontaneous fermentations of two grape varietals at two commercial wineries. Yeast isolates collected from fermentations were identified to the strain level using microsatellite analysis. Commercial strains made up the majority (64-98%) of the S. cerevisiae strains isolated during fermentation, and most of these commercial strains were used as inoculants by their respective wineries. Different SO 2 additions were found to significantly alter S. cerevisiae strain compositions at both wineries (p≤0.002). The results of this study demonstrate that initial SO 2 addition significantly alters the S. cerevisiae strain composition in spontaneous fermentations, and highlights the dominance of commercial strains in commercial winery environments. Because different yeast strains are known to produce different chemical and sensory profiles, our findings have important implications for winemakers. In addition, adding different concentrations of SO 2 may be a way for winemakers to manage or control the strain composition during spontaneous fermentations., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Soil depth partitioning is thought to promote the diversity of ectomycorrhizal (EM) fungal communities, but little is known about whether it is controlled by abiotic or biotic factors. In three bioassay experiments, we tested the role of vertical soil heterogeneity in determining the distributions and competitive outcomes of the EM sister species Rhizopogon vinicolor and Rhizopogon vesiculosus. We planted Pseudotsuga menziesii seedlings into soils that were either a homogenized mix of upper and lower depths or vertically stratified combinations mimicking natural field conditions. We found that both species colonized the upper or lower soil depths in the absence of competition, suggesting that their distributions were not limited by abiotic edaphic factors. In competition within homogeneous soils, R. vesiculosus completely excluded colonization by R. vinicolor, but R. vinicolor was able to persist when soils were stratified. The amount of colonization by R. vinicolor in the stratified soils was also significantly correlated with the number of multilocus genotypes present. Taken together, our findings suggest that the differential vertical distributions of R. vinicolor and R. vesiculosus in natural settings are probably attributable to competition rather than edaphic specialization, but that soil heterogeneity may play a key role in promoting EM fungal diversity., (© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)

Rhizopogon vesiculosus and Rhizopogon vinicolor are sister species of ectomycorrhizal fungi that associate exclusively with Douglas-fir (DF). They form tuberculate mycorrhizas and they can be easily distinguished using molecular tools. We are not aware of studies relating their relative abundance in forests with different age classes. Our objective was to determine whether a change in the number or relative abundance of R. vesiculosus and R. vinicolor tubercules and genotypes was related to a change in the percent of DF in a regenerating phase (<50 years old). R. vesiculosus and R. vinicolor were located by excavating tuberculate mycorrhizas from the forest floor. A DNA Alu1 digest was used to distinguish between the two species. Microsatellite markers were used to identify genotypes. The number of R. vesiculosus tubercules correlated positively with an increasing proportion of DF in a regenerating phase, while the number of R. vinicolor tubercules was similar across all forest age structures. The number of R. vesiculosus genotypes did not correlate with forest age structure, whereas the number of R. vinicolor genotypes showed a negative relationship with an increasing proportion of DF in a regenerating phase. When the numbers of R. vesiculosus tubercules and genotypes were expressed as a relative abundance of the two species, there was a positive correlation with an increasing proportion of DF in a regenerating phase for both genotypes and tubercules. Our results suggest that the degree of DF regeneration or ecosystem factors related to DF regeneration affect the population dynamics of R. vesiculosus and R. vinicolor differently.

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.

Inoculated fermentations are practiced in most wine regions of the world. This type of fermentation involves adding a commercial Saccharomyces cerevisiae strain as an inoculant. It is often assumed that the inoculant maintains dominance throughout the fermentation; however, sometimes commercial or indigenous yeasts, which were not intentionally added, end up as the dominant yeast in the winery fermentation. The aim of this study was to compare implantation/persistence of inoculants among three Canadian wineries (Quails' Gate, Cedar Creek, and Road 13 wineries). In 2010, three inoculated fermentation tanks at each of three wineries were sampled at four stages of fermentation (pre-inoculation, early, mid, and end). In addition, results from the end stage of fermentation, from two of the three wineries, were compared among different vintages (resulting in a 4-year comparison at Quails' Gate winery and a 2-year comparison at Cedar Creek winery). Strains of S. cerevisiae were discriminated by microsatellite analysis and identified using commercial microsatellite databases, whereas DNA sequencing was used to identify non-Saccharomyces. The percent implantation/persistence of the inoculum was significantly lower at Quails' Gate and Cedar Creek wineries as compared with the Road 13 winery in the 2010 vintage. Relatively low persistence of the inoculum at Quails' Gate winery was also found in the 2009 vintage, but low values were not found at Quails' Gate winery in 2011 and 2012 or at Cedar Creek winery in 2012. In all tanks having <80% relative abundance of the inoculant, the commercial strain (Lalvin ICV-D254®/Fermol® Premier Cru) was the dominant or co-dominant yeast. Our findings highlight year-to-year variation in inoculum implantation/persistence and the idea that unless strain typing of S. cerevisiae is conducted at the winery, there are no obvious fermentation factors that would indicate a relatively low inoculum implantation/persistence., (Copyright © 2014. Published by Elsevier B.V.)

Wildfire severity in forests is projected to increase with warming and drying conditions associated with climate change. Our objective was to determine the impact of wildfire and clearcutting severity on the ectomycorrhizal fungal (EMF) community of Douglas-fir seedlings in the dry forests of interior British Columbia, Canada. We located our study within and surrounding the area of the McLure fire (August 2003). We hypothesized that disturbance would affect EMF community assembly due to reductions in fungal inoculum. Five treatments representing a range of disturbance severities were compared: high severity burn, low severity burn, screefed clearcut (manual removal of forest floor), clearcut, and undisturbed forest. EMF communities in the undisturbed forest were more complex than those in all disturbance treatments. However, aspects of community assembly varied with disturbance type, where the burn treatments had the simplest communities. After 4 months, regenerating seedlings in the burn treatments had the lowest colonization, but seedlings in all treatments were fully colonized within 1 year. EMF communities were similar among the four disturbance types, largely due to dominance of Wilcoxina throughout the study period. However, forest floor retention influenced community assembly as the EMF in the clearcut treatment, where forest floor was retained, had levels of diversity and richness comparable to the undisturbed forest. Overall, the results suggest that increasing forest floor disturbance can alter EMF community assembly in the first year of regeneration. A correlation between poorly colonized seedlings and seedling productivity also suggests a role for productivity in influencing community assembly.

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., (© 2012 Blackwell Publishing Ltd.)

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.

*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.

Mycorrhizal networks (MNs) are fungal hyphae that connect roots of at least two plants. It has been suggested that these networks are ecologically relevant because they may facilitate interplant resource transfer and improve regeneration dynamics. This study investigated the effects of MNs on seedling survival, growth and physiological responses, interplant resource (carbon and nitrogen) transfer, and ectomycorrhizal (EM) fungal colonization of seedlings by trees in dry interior Douglas-fir (Pseudotsuga menziesii var. glauca) forests. On a large, recently harvested site that retained some older trees, we established 160 isolated plots containing pairs of older Douglas-fir "donor" trees and either manually sown seed or planted Douglas-fir "receiver" seedlings. Seed- and greenhouse-grown seedlings were sown and planted into four mesh treatments that served to restrict MN access (i.e., planted into mesh bags with 0.5-, 35-, 250-microm pores, or without mesh). Older trees were pulse labeled with carbon (13CO2) and nitrogen (15NH4(15)NO3) to quantify resource transfer. After two years, seedlings grown from seed in the field had the greatest survival and received the greatest amounts of transferred carbon (0.0063% of donor photo-assimilates) and nitrogen (0.0018%) where they were grown without mesh; however, planted seedlings were not affected by access to tree roots and hyphae. Size of "donor" trees was inversely related to the amount of carbon transferred to seedlings. The potential for MNs to form was high (based on high similarity of EM communities between hosts), and MN-mediated colonization appeared only to be important for seedlings grown from seed in the field. These results demonstrate that MNs and mycorrhizal roots of trees may be ecologically important for natural regeneration in dry forests, but it is still uncertain whether resource transfer is an important mechanism underlying seedling establishment.

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.

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.

Ectomycorrhizal (ECM) fungal communities of Douglas-fir (Pseudotsuga menziesii) and paper birch (Betula papyrifera) were studied along a chronosequence of forest development after stand-replacing disturbance. Previous studies of ECM succession did not use molecular techniques for fungal identification or lacked replication, and none examined different host species. Four age classes of mixed forests were sampled: 5-, 26-, 65-, and 100-yr-old, including wildfire-origin stands from all four classes and stands of clearcut origin from the youngest two classes. Morphotyping and DNA sequences were used to identify fungi on ECM root tips. ECM fungal diversities were lower in 5-yr-old than in older stands on Douglas-fir, but were similar among age classes on paper birch. Host-specific fungi dominated in 5-yr-old stands, but host generalists were dominant in the oldest two age classes. ECM fungal community compositions were similar in 65- and 100-yr-old stands but differed among all other pairs of age classes. Within the age range studied, site-level ECM fungal diversity reached a plateau by the 26-yr-old age class, while community composition stabilized by the 65-yr-old class. Simple categories such as 'early stage', 'multi stage', and 'late stage' were insufficient to describe fungal species' successional patterns. Rather, ECM fungal succession may be best described in the context of stand development.

We conducted greenhouse experiments using Douglas-fir (Pseudotsuga menziesii var. glauca) seedlings where chemical methods (fungicides) were used to prevent ectomycorrhizal colonization of single seedlings or physical methods (mesh barriers) were used to prevent formation of mycorrhizal connections between neighboring seedlings. These methods were chosen for their ease of application in the field. We applied the fungicides, Topas (nonspecific) and Senator (ascomycete specific), separately and in combination at different concentrations and application frequencies to seedlings grown in unsterilized forest soils. Additionally, we assessed the ability of hyphae to penetrate mesh barriers of various pore sizes (0.2, 1, 20, and 500 microm) to form mycorrhizas on roots of neighboring seedlings. Ectomycorrhizal colonization was reduced by approximately 55% with the application of Topas at 0.5 g l(-1). Meshes with pore sizes of 0.2 and 1 microm were effective in preventing the formation of mycorrhizas via hyphal growth across the mesh barriers. Hence, meshes in this range of pore sizes could also be used to prevent the formation of common mycorrhizal networks in the field. Depending on the ecological question of interest, Topas or the employment of mesh with pore sizes <1 microm are suitable for restricting mycorrhization in the field.

The effects on the ectomycorrhizal fungal community of clearcut logging, which is used to harvest millions of hectares of ectomycorrhizal forest annually, has been studied for a number of years. Here, we review current knowledge of inoculum sources for ectomycorrhizal fungi in forests and then re-examine earlier studies of ectomycorrhizas on young trees in regenerating stands. We conclude that, taken separately from the effects of site preparation, the major impact of clearcut logging is to change the species composition of the ectomycorrhizal fungal community rather than to reduce the percentage of roots colonized. A thorough examination of site preparation treatments suggests that the changes in fungal species composition are driven by changes in the biology and chemistry of the soil environment after clearcutting as much as they are by loss or change in fungal inoculum. This is an important conclusion because it implies that these new ectomycorrhizal fungal communities are better adapted to the new conditions than the ones in the forest would have been. The shift in fungal species composition and diversity will have implications for seedling establishment and competition. The effects of individual fungi or diverse assemblages of fungi on seedling growth, and effects of changes in the ability of young trees to associate with a common mycelium are discussed. Contents Summary 399 I. Introduction 400 II. Population biology and inoculum potential of ectomycorrhizal fungi 401 III. Ectomycorrhiza development on seedlings regenerating after clearcut logging 402 IV. Which is the most important factor driving changes in the ECM fungal community after clearcut logging: inoculum loss or change in the below-ground environment? 406 V. Possible consequences for regenerating stands of species shifts in ectomycorrhizal fungi 414 VI. Conclusions 416.

Interspecific C transfer was studied in laboratory microcosms containing pairs of 6-month-old Betula papyrifera Marsh, and Pseudotsuga menziesii (Mirb.) Franca seedlings growing in individual, root-restrictive (28μm pore size) pouches filled with field soil. Interspecific transfer was examined by reciprocal labelling of seedlings with 13 CO 2(gas) and 14 CO 2(gas) . At the time of labelling, the root zones of ectomycorrhizal (EM) B. papyrifera and P. menziesii were interconnected by an extensive network of EM mycelium. Carbon transferred through EM connections was distinguished from that through soil pathways by comparing microcosms where interconnecting hyphae were left intact vs. those where they were severed immediately before labelling. Transfer was bidirectional, and represented 5 % of total isotope uptake by both B. papyrifera and P. menziesii together. P. menziesii received on average 50% more 14 C and 66% more 13 C from paper birch than vice versa, however, differences between species were not statistically significant. Neither net nor bidirectional transfer differed between severing treatments, leaving in question the relative importance of EM hyphae versus soil transfer pathways. The tendency for P. menziesii to receive more isotope than B. papyrifera corresponded with a 10-fold greater net photosynthetic rate per seedling and two-fold greater foliar N concentration of B. papyrifera than P. menziesii.

The benefit to a fungus by a mycorrhizal association is that it gains carbon from its host. A benefit to a host is usually a nutritional one, but any resulting increase in dry weight may be counteracted by the carbon consumed by the fungus. The carbon costs of mycorrhizal fungi have been calculated using 14 C pulse-chase experiments in the laboratory or by estimating turnover rates in the field. Both of these techniques have their limitations, but estimates have been remarkably consistent amongst most laboratory studies. Carbon demands of the fungus may not reduce theoretical growth enhancement in plants which are sink-limited but would be expected to do so iii source-limited plants. A model of carbon use efficiency is developed based on the economic concepts of cost and benefit. Efficiency is defined in terms of carbon gained via the growth response to infection, and the carbon expended supporting the fungus. Practical considerations of measuring carbon allocation, and calculating carbon use efficiency are discussed. In an experiment on Salix viminalis L., colonized by Thelephora terrestris (Ehrh.) Fr., the carbon use efficiency calculated by this method was on overage 85% based on shoot tissue production, and 95% based on whole plant production.

The flow of carbon from plant to fungus in ectomycorrhizal associations has not been well quantified. The objective of this study was to use 14 C to quantify the increase in fixed carbon translocated below ground in ectomycorrhizal relative to non-mycorrhizal willow (Salix viminalis L, Bowles hybrid). Rooted cuttings were inoculated with Thelephora terrestris (Ehrh). Fr. or left non-mycorrhizal. Non-mycorrhizal plants were grown at the same (4 mg kg -l bicarbonate-extractable P) (NM-P) or at a higher (21 mg kg -1 ) soil P concentration (NM +P), one at which the non-mycorrhizal plants were similar in size to the mycorrhizal (M-P) plants. At 41, 51, 76 and 89 days after planting, the shoots were exposed to a pulse of 14 CO 2 . Plants were harvested after a 202 h chase period. The 14 Cactivity was quantified in live fractions: shoot tissue, shoot respiration, 'root' tissue (= roots plus fungi), 'root' respiration (= CO 2 released below ground) and soil. Of the total 14 C detected in these five fractions, M-P plants allocated from 3.9% (harvest 1) to 11.5% (harvest 4) more to the below-ground fractions ('root' tissue, soil and 'root' respiration), than did the NM-P plants. Differences between NM+P and M-P plants were only half of those above (3.1 % and 4.4% at harvests 2 and 3, respectively, compared to 6.4 % and 7.4%, respectively for the difference between NM-P and M-P). Correction for differences in root/shoot ratio between M-P and NM-P plants eliminated the observed differences in carbon distribution only at the first three harvests. There was no evidence for increased 'root' respiration fates or rhizodeposition being responsible for the increased carbon diverted below ground by M-P plants.

Ectomycorrhizas of Pseudotsuga menziesii (Mirb.) Franco were synthesized in pure culture with Cenococcum geophilum Fr., Laccaria bicolor (Maire) Orton, Rhizopogon vinicolor Smith, or Suillus lakei (Murr.) Smith and Thiers. The mycorrhizal plants were grown in sterile vessels with peat/vermiculite plus 14 C labelled hemicellulose, cellulose, Douglas fir needles, or humic polymers as an organic substrate. The sum of the 14 C respired and that found in the seedling after 240 d of growth was used to quantify decomposition of the labelled substrates. All mycobionts released at least 20-30% of the 14 C in hemicellulose and all were able to release 14 C from cellulose and needles, escept L. buolttr, which failed to release 14 C from needles. S. lakei released the largest amount of 14 C from cellulose. C. geophilum, S. lakei and R. filticolor released similar amounts of 14 C from needles. Generally, substrates could be ranked in terms of ease of breakdown by the ectomycorrhizal fungi as follows: humic polymers < needles < cellulose < hemicellulose.

Seedlings of Pinus Ponderosa Dougl, ex. Laws were grown in root observation containers. They were inoculated with either Hebeloma crustuliniforme (Bull.) Quel. or Laccaria bicolor (Maire) Orton or left uninoculated (control). Oil a monthly basis starting at the eighth month of a 12-month growing period, roots (mycorrhizal root tips from inoculated plants, non-mycorrhizal from control plants) were traced onto acetate sheets. Each root tip was classified as light, 'intermediate' or 'dark' in colour. Roots initiated between months eight and nine were monitored for the next 90 d. All root tips progressed from light-brown to dark-brown to black for all three treatments. Hebeloma crustuliniforme, and to a lesser extent L. bicolor, retarded this progression relative to the control. At the end of 12 months, seedlings were labelled with 14 CO 2 to determine the effects of ectomycorrhizal inoculation on carbon supply to roots of the different morphological categories. The amount of 14 C in 'light'L. bicolor and H. crustuliniforme mycorrhizas was 2.3 and 1.8 times greater, respectively, than that in 'light' control root tips. The amount of 14 C in mycorrhizas of the inoculated treatments and, to a lesser extent, roots of the control seedlings decreased as they progressed from 'light' to 'dark'. It is concluded that pondersa pine seedlings continue to allocate photosynthate to morphologically older roots, perhaps to meet maintenance requirements or to supply carbon for growth and metabolism of extra-matrical hyphae. Such allocation may enhance root longevity, which would have an important influence on tree, forest and soil carbon budgets.

One way of viewing a mycorrhizal symbiosis is as a balance between the nutritional 'benefits' and carbon 'costs' to the phytobiont. Phosphorus acquisition efficiency (the amount of phosphorus taken up per unit of carbon allocated belowground) can be used as an indicator of this balance. In this study, phosphorus uptake and belowground carbon allocation were measured using ectomycorrhizal (M) (Thelephora terrestris (Ehrh.) Fr.) and non-mycorrhizal (NM) Salix viminalis L. cv. Bowles Hybrid. Following 50, 60, 85 or 98 d of growth in a gamma-irradiated soil/sand mixture containing 4 mg bicarbunate-extractable P kg -1 , seven randomly-selected cuttings of each treatment were harvested and their P contents determined. Nine d prior to each harvest, the three median plants from the group of seven were pulse labelled with 14 C to determine the relative allocation of C aboveground and belowground. Mycorrhizal colonization of willow caused a two-fold increase in growth owing to substantially higher P uptake. Phosphorus inflow rates were almost three times as high for M root systems as for NM root systems over the interval up to the first harvest (3.2 × 10 -12 and 1.2 × 10 12 mol m -1 s-1, respectively). Over the interval from 50 to 98 d, inflows into M plants were 50% higher than into NM plants (1.4 × 10 12 and 0.9 × 10 -13 mol m -1 s -1 respectively). The M plants allocated about 25 times as much carbon belowground as the NM plants for both periods. The P acquisition efficiency was higher in M than in NM plants during the first interval (16% and 40% higher using two different calculation methods), whereas during the second interval it was higher in NM than in M plants (33% and 44% higher using the two different methods). Thus, ectomycorrhizas can be very effective in supplying P to their hosts even at an early stage of infection. Furthermore, it is suggested that a temporal separation exists in the maximal fluxes of P and C between the fungus and the host of the mycorrhizal association. The results are discussed in the context of the nutrient requirements and carbon economies of field-grown woody plants.

There is much circumstantial evidence for a role of increased P uptake in the growth response of plants to ectomycorrhizas. Full response curves with and without mycorrhizal infection along a P gradient in soil are, however, required to test this hypothesis fully. In this experiment, rooted cuttings of Salix viminalis L. cv. Bowles Hybrid were grown in a 1:2 mixture by volume of gamma-irradiated soil and sterile sand, with bicarbonate-extractable P concentrations of 4, 6, 10, 21, 60 or 90 mg P kg -1 . The cuttings were inoculated by mixing peat/vermiculite spawn of Laccaria proxima (Boud.) Pat., or Thelephora terrestris (Ehrh.) Fr., or autoclaved spawn 1: 5 by volume with the soil: sand mixture. The plants showed a positive growth response to mycorrhizal infection by either fungus at the two lower P levels, and to L. proxima only at 10 mg P kg -1 . At 21 mg kg -1 and above, infection was reduced and neither mycorrhizal inoculation nor further P additions caused significant growth increases. These results imply that the growth responses to ectomycorrhizas in this experiment were solely due to increases in P uptake. Cuttings infected with L. proxima tended to be larger than those infected by T. terrestris. Estimates of percent mycorrhizal infection did not differ between the fungi at the lower P levels. However, overall production of extramatrical hyphae per gram of soil was highest in soil inoculated with L. proxima. At 10 mg P kg -1 the length of L. proxima hyphae per unit length of mycorrhizal root, P uptake per unit root weight, and total P content in plants infected with L. proxima were significantly higher than for T. terrestris. This study confirms that increased P uptake can be an important component of growth stimulation by ectomycorrhizas. It also presents the first quantification of extramatrical ectomycorrhizal hyphae in soil and suggests a role for them in the growth response.

Ponderosa pine seedlings were inoculated with Hebeloma crustuliniforme either in growth pouches before they were transplanted to root-mycocosms (P seedlings), or at the time of transfer to root-mycocosms (V seedlings). Uninoculated seedlings served as controls (U seedlings). The use of root-mycocosms allowed examination of portions of hyphae separate from roots and rooting substrate but still in symbiosis with the host. The results thus provided a quantitative basis for estimating hyphal mass and carbon allocation to extramatrical hyphae. The amount of (14)CO(2) fixed after a 2-h exposure was greatest for P seedlings and least for uninoculated seedlings. Four and nine days after exposure, (14)C content was greatest in uninoculated seedlings and least in inoculated seedlings. In isotope distribution and dry mass accumulation, V seedlings were more similar to U than to P seedlings. Calculated on a dry weight basis, the allocation of isotope to mycelium suggested that extramatrical hyphae of P seedlings were a stronger sink for carbon than extramatrical hyphae of V seedlings. Differences in inoculation methods resulted in differences in carbon allocation and physiology of extramatrical hyphae that could affect seedling establishment and survival. Seedlings inoculated by one method cannot serve as surrogates for mycorrhizal seedlings produced by other inoculation techniques.

Short-term effects of ozone (O(3)) on phyllosphere fungi were studied by examining fungal populations from leaves of giant sequoia (Sequoiadendron giganteum (Lindl.) Buchholz) and California black oak (Quercus kelloggii Newb.). Chronic effects of both O(3) and sulfur dioxide (SO(2)) were studied by isolating fungi from leaves of mature Valencia orange (Citrus sinensis L.) trees. In this chronic-exposure experiment, mature orange trees were fumigated in open-top chambers at the University of California, Riverside, for 4 years with filtered air, ambient air plus filtered air (1:1), ambient air, or filtered air plus SO(2) at 9.3 parts per hundred million. Populations of Alternaria alternata (Fr.) Keissler and Cladosporium cladosporioides (Fres.) de Vries, two of the four most common fungi isolated from orange leaves, were significantly reduced by chronic exposure to ambient air. In the short-term experiments, seedlings of giant sequoia or California black oak were fumigated in open-top chambers in Sequoia National Park for 9 to 11 weeks with filtered air, ambient air, or ambient air plus O(3). These short-term fumigations did not significantly affect the numbers of phyllosphere fungi. Exposure of Valencia orange trees to SO(2) at 9.3 parts per hundred million for 4 years reduced the number of phyllosphere fungi isolated by 75% compared with the number from the filtered-air treatment and reduced the Simpson diversity index value from 3.3 to 2.5. A significant chamber effect was evident since leaves of giant sequoia and California black oak located outside of chambers had more phyllosphere fungi than did seedlings within chambers. Results suggest that chronic exposure to ambient ozone or SO(2) in polluted areas can affect phyllosphere fungal communities, while short-term exposures may not significantly disturb phyllosphere fungi.