Societal Impact Statement Forest management for wood products involves the conversion of primary forests to younger plantations. This has an immediate impact on biota such as ectomycorrhizal fungi, a diverse symbiotic fungal community. Alternatives to large clearcuts may be one way to mitigate harvesting effects. We tested this premise by examining ectomycorrhizal fungal communities across forest edges in 1‐ha openings at nearly three decades postharvest. We found reductions in species richness and shifts in community composition were still evident, illustrating how recovery was limited by the open canopies of younger trees. Sustainable management of ectomycorrhiza may require further conservation strategies, particularly green‐tree retention. Summary Ectomycorrhizal fungi (EMF) are sensitive to stand‐removing disturbances; diverse EMF communities, especially rarer fungi typical of old‐growth stands, may take a century to re‐establish. Proximity to mature forests via small cutovers could accelerate postharvest EMF community recovery, thereby supporting sustainable forest management. Alternatively, forest edges may impose a fragmentation effect, which could eventually negatively influence EMF taxa in the remaining forest. We compared both succession and fragmentation patterns across 1‐ha openings 27 years postharvest in a high‐elevation coniferous forest. We sampled 90‐m transects across forest edges of nine openings, using long‐read metabarcoding of EMF from mycorrhizal root tips, and Sanger sequencing of epigeous sporocarps to assess EMF community response. A timber harvesting effect on root tip EMF communities was still evident, with differences among species assemblages between the opening, edge zone, and interior forest habitats. EMF richness of root tips in openings declined by 27%, on average, beginning at 10 m from the forest edge. While EMF richness on roots was consistent across forest plots, fruiting was curtailed within forest edge zones, alongside some reductions in forest‐dependent taxa. Overall, the potential benefits of small openings were apparently constrained by environmental factors that delay the re‐establishment of late‐seral EMF species under open canopies. The negative effect of edges on EMF reproduction and some taxa abundance in the forest adjacent to openings is relevant when designing green‐tree retention forestry systems, suggesting medium to large patches of trees may reduce fragmentation stress and better sustain an array of late‐seral fungi.

With wildfires reaching unprecedented levels of severity, size and frequency, their impact on soil microbial communities is an important concern. Commencing just weeks following a wildfire in a Douglas-fir-Ponderosa pine forest, we collected surface and mineral soil samples four times over 10 months from four severity levels to better understand the effect of wildfire severity on fungi and their guilds. At medium severity and above, there was a decrease in fungal diversity and a shift in community composition in both layers, while the fungal community from soils burnt by low severity fires remained similar to unburnt soils. Although the richness of putative saprotrophic and pathotrophic fungi in burnt microplots returned to levels comparable to that in the unburnt microplots within weeks of burning, the richness of symbiotrophic, including ectomycorrhizal fungi in burnt plots, did not. The DNA of many putative pyrophilous fungi peaked in estimated abundance within weeks after the fire, even though fruiting was not observed until the following spring. Index descriptors: pyrophilous fungi; wildfire severity; ITS; metabarcoding; Pseudotsuga menziesii var. glauca; fungal community; disturbance; succession.

Each wine growing region hosts unique communities of indigenous yeast species, which may enter fermentation and contribute to the final flavour profile of wines. One of these species, Saccharomyces uvarum, is typically described as a cryotolerant yeast that produces relatively high levels of glycerol and rose-scented volatile compounds as compared with Saccharomyces cerevisiae, the main yeast in winemaking. Comparisons of fermentative and chemical properties between S. uvarum and S. cerevisiae at the species level are relatively common; however, a paucity of information has been collected on the potential variability present among S. uvarum strains. The objective of this study was to compare the fermentation kinetics and production of volatile compounds between indigenous and commercial Saccharomyces strains at different temperatures. We compared laboratory-scale fermentation of Chardonnay juice at 15 °C and 25 °C for 11 Saccharomyces yeast strains (six indigenous S. uvarum, one commercial S. uvarum, one indigenous S. cerevisiae and three commercial S. cerevisiae). Fermentation kinetics and the production of volatile compounds known to affect the organoleptic properties of wine were determined. The indigenous S. uvarum strains showed comparable kinetics to commercially sourced strains at both temperatures. Volatile compound production among the strains was more variable at 15 °C and resulted in unique chemical profiles at 15 °C as compared with 25 °C. Indigenous S. uvarum strains produced relatively high levels of 2-phenylethyl acetate and 2-phenylethanol, whereas these compounds were found at much lower levels in fermentations conducted by commercial strains of both S. cerevisiae and S. uvarum. Production of glycerol by indigenous S. uvarum strains did not differ from commercial strains in this study. Our findings demonstrate that indigenous strains of S. uvarum show functional variation among themselves. However, when compared with commercial S. cerevisiae and S. uvarum strains, they have comparable fermentation kinetics but unique volatile compound profiles, especially at low fermentation temperatures.

Background When identifying transcript abundance in response to treatment, accurate quantification is critical, especially when examining subtle differences in expression. In particular, data normalization is necessary to account for differences among samples including those associated with RNA quantity and quality. Due to the capacity of droplet digital PCR to absolutely quantify the copy number of the target gene in a given sample, normalization, such as the use of an internal control gene, has not customarily been considered obligatory. Decades of quantitative PCR research have shown, however, that the use of endogenous controls undoubtedly aid in correcting sample variability. With our limited knowledge of gene function in many fungi, typical ‘housekeeping genes’ commonly used as internal references may not be relevant in these organisms. This study aimed to identify and validate suitable reference genes for transcript abundance studies in Oidiodendron maius , a globally distributed, model ericoid mycorrhizal fungus. Results A shortlist of 251 non-differentially expressed genes was generated from RNA-Seq analyses of O. maius grown on three different carbon sources or in symbiosis with Vaccinium myrtillus . Subsequently, a set of criteria (stable expression, valid annotation and relatively high expression) was applied to select three candidate reference genes. These three genes were validated across a further eleven carbon sources using ddPCR and the application of geNorm and NormFinder stability analysis algorithms. Expression stability analysis of three genes - EfTu , vma , and sar - confirmed their reliability as internal references; the geometric mean of their expression values demonstrated the highest stability as a normalization factor. Conclusions We propose the use of the geometric mean of O. maius genes EfTu , vma and sar as a reference tool to normalize RNA expression in ddPCR assays. These newly selected and validated reference genes will increase reliability and reproducibility when studying transcriptional responses of O. maius at different developmental stages and/or under a range of physiological conditions. In addition, the list of 251 non-differentially expressed genes can serve as a valuable resource for selecting reference genes for related experiments and enhances the limited information available on O. maius .

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.

Saccharomyces cerevisiae is usually the dominant yeast in winemaking. However, other yeast species such as Saccharomyces uvarum are also able to conduct and complete alcoholic fermentation. S. uvarum is a cryotolerant yeast that has been found dominating low-temperature fermentations at commercial wineries around the world, but little research has been conducted to investigate its competitive abilities relative to commercial S. cerevisiae strains. Here, controlled Chardonnay fermentations were conducted with a commercial S. cerevisiae strain and an indigenous S. uvarum strain inoculated at different initial ratios and two different temperatures. Both strains fermented well at 24°C, and both strains fermented slowly at 15°C. S. cerevisiae was more competitive than S. uvarum when inoculated at an equal or greater ratio. However, S. uvarum was better able to grow and compete with S. cerevisiae at a lower fermentation temperature and, when inoculated at an equal ratio, was able to maintain a 25% relative abundance throughout fermentation, contributing to the volatile-compound profile of the wine. Volatile-compound production was distinct for the two yeast strains, with S. uvarum producing generally higher amounts of volatile compounds, especially at the lower fermentation temperature, including higher amounts of 2-phenylethyl acetate (honey/spice/floral) and ethyl 2-methyl butanoate (apple/strawberry). When both strains co-existed in fermentation, the resulting volatile profile was unique in that it differed from either single-strain fermentation. This study is the first to test co-inoculation of S. cerevisiae and S. uvarum at different ratios and different fermentation temperatures and highlights the potential for the use of indigenous S. uvarum strains in isolation or in combination with S. cerevisiae to produce unique, quality wines.

Background and Aims During winemaking, alcoholic and malolactic fermentations are usually conducted by strains ofSaccharomyces cerevisiaeandOenococcus oeni, respectively. Although most commercial wines are produced via inoculation with both yeast and bacteria, uninoculated fermentations can increase microbial diversity, leading to more complex wines. In this study, Chardonnay must was subjected to a combination of inoculation treatments withS. cerevisiaeandO. oeniin order to investigate how yeast and bacterial inoculation may impact the microbial communities of these wines. Methods and Results Yeast and bacterial community dynamics, andS. cerevisiaeandO. oenipopulations, were monitored at different fermentation stages. Yeast inoculation practices altered the yeast community andS. cerevisiaestrains; wines left uninoculated had a higher diversity ofS. cerevisiaestrains and contained an increased abundance ofTorulaspora delbrueckii. The inoculatedO. oenistrain dominated the treatments to which it was added, but the uninoculated wines contained uniqueO. oenipopulations, including potentially indigenous dominant strains. Conclusions Uninoculated wines contained a greater diversity of yeast and bacteria than inoculated wines, including more indigenous/non-commercial species and strains. Significance of the Study These results highlight the importance of identifying the entire microbial community in fermentations, at both the species and strain level.

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.

Sulfur dioxide (SO2) is an antioxidant and antimicrobial agent used in winemaking. Its effects on spoilage microorganisms has been studied extensively, but its effects on commercial Saccharomyces cerevisiae strains, the dominant yeast in winemaking, require further investigation. To our knowledge, no previous studies have investigated both the potential SO2 resistance mechanisms of commercial yeasts as well as their production of aroma-active volatile compounds in response to SO2. To study this, fermentations of two commercial yeast strains were conducted in the presence (50 mg/L) and absence (0 mg/L) of SO2. Strain QA23 was more sensitive to SO2 than Strain BRL97, resulting in delayed cell growth and slower fermentation. BRL97 exhibited a more rapid decrease in free SO2, a higher initial production of hydrogen sulfide, and a higher production of acetaldehyde, suggesting that each strain may utilize different mechanisms of sulfite resistance. SO2 addition did not affect the production of aroma-active volatile compounds in QA23, but significantly altered the volatile profiles of the wines fermented by BRL97.

Sulfur dioxide (SO2) has been used in conjunction with winemaking for centuries, and the practice of burning sulfur candles inside wine vessels to preserve the wine dates back to ancient Roman times. Today, the use of sulfur dioxide is ubiquitous throughout the world of winemaking, and many consider it a non-negotiable additive, acting as both an antioxidant and an antimicrobial agent. SO2 is almost always added at bottling to help preserve the wine, and is often added at crush to remove potential spoilage microbes. However, a growing consumer base has pushed for the production of wines with fewer chemical additives, including wines with lower levels of SO2. High levels of SO2 in wines can have a negative effect on health (in vulnerable populations) and on wine quality. While there are alternatives on the market, including ultraviolet light, colloidal silver complex, and dimethyl dicarbonate, none are as effective as SO2 as both an antioxidant and antimicrobial agent.

Modern day winemaking often involves the addition of sulfur dioxide (SO2) at crush to act as both an antioxidant and an antimicrobial agent. While the effects of SO2 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 SO2 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 SO2 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 SO2 had been added. Two key wine sensory attributes (citrus aroma and pome fruit flavor) differed among the SO2 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 SO2 additions during the crush period.

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.

There is a lack of knowledge about the composition of Saccharomyces cerevisiae strains in spontaneous fermentations of Pinot Noir and Chardonnay cultivars. The objectives were to determine the relative abundance of indigenous and commercial S. cerevisiae strains in spontaneous fermentations at three wineries from the two cultivars and to compare the composition of the S. cerevisiae strains between cultivars and wineries. Three fermentation vessels were sampled at three stages of fermentation for each cultivar at each winery. Isolates were identified to the strain level using seven microsatellite loci. Commercial S. cerevisiae strains were isolated at a frequency higher than that of the indigenous strains at each winery for both cultivars. The composition of S. cerevisiae strains was different for each cultivar and at each winery. Our results illustrate the clear influence of inoculated commercial active dry yeast strains on the composition of S. cerevisiae strains in spontaneous fermentations at wineries conducting both inoculated and spontaneous fermentations.

Previous winery-based studies showed the strains Lalvin® RC212 (RC212) and Lalvin® ICV-D254 (D254), when present together during fermentation, contributed to >80% relative abundance of the Saccharomyces cerevisiae population in inoculated and spontaneous fermentations. In these studies, D254 appeared to out-compete RC212, even when RC212 was used as the inoculant. In the present study, under controlled conditions, we tested the hypotheses that D254 would out-compete RC212 during fermentation and have a greater impact on key fermentation-derived chemicals. The experiment consisted of four fermentation treatments, each conducted in triplicate: a pure culture control of RC212; a pure culture control of D254; a 1:1 co-inoculation ratio of RC212:D254; and a 4:1 co-inoculation ratio of RC212:D254. Strain abundance was monitored at four stages. Inoculation ratios remained the same throughout fermentation, indicating an absence of competitive exclusion by either strain. The chemical profile of the 1:1 treatment closely resembled pure D254 fermentations, suggesting D254, under laboratory conditions, had a greater influence on the selected sensory compounds than did RC212. Nevertheless, the chemical profile of the 4:1 treatment, in which RC212 dominated, resembled that of pure RC212 fermentations. Our results support the idea that co-inoculation of strains creates a new chemical profile not seen in the pure cultures. These findings may have implications for winemakers looking to control wine aroma and flavor profiles through strain selection.

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.

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.